Estrogen receptor modulators

ABSTRACT

Isoxazole estrogen receptor agonist and antagonist compounds having unexpected and surprising activity in modulating estrogen receptor activity are described. In addition, methods and compositions for treating or preventing estrogen receptor-mediated disorders are disclosed. The compounds, methods, and compositions of the invention have utility in preventing or treating estrogen receptor-mediated disorders such as osteoporosis, breast and endometrial cancers, atherosclerosis, and Alzheimer&#39;s disease.

1 CROSS REFERENCE TO RELATED U.S. PATENT APPLICATIONS

This appln is a div of Ser. No. 09/369,748 Aug. 6, 1999, U.S. Pat. No.6,262,098, which claims benefit of provisional U.S. patent applicationserial No. 60/095,773, Aug. 7, 1998.

2 BACKGROUND OF THE INVENTION

2.1 Field of the Invention

The present invention relates to compounds that have biological activitywith respect to estrogen receptors and to the use of such compounds totreat diseases and disorders related to estrogen receptor activity. Moreparticularly, the present invention provides selective estrogen receptormodulators (“SERMs”). The present invention therefore relates to thefields of medicine, medicinal chemistry, biochemistry, andendocrinology.

2.2 Background

Estrogen is a hormone critical to normal human development and function.Although estrogen is the predominant “sex hormone” in women, in whomestrogen controls the development of female sex characteristics and thedevelopment and function of the reproductive system (Berkow, Beers etal. 1997), it is also found in men (Gustafsson 1998). Women produceestrogen primarily in the ovaries; however, estrogen affects a varietyof physiological functions in women including body temperatureregulation, maintenance of the vaginal lining, and preservation of bonedensity (Jordan 1998). In addition, estrogen provides additional effectsthat are related to its ability to modulate production of cholesterol inthe liver, as demonstrated by the reduced occurrence of atherocsclerosisin women compared to men due in part to the reduction of low-densitylipoprotein (“LDL”) (Jordan 1998). Estrogen has also been implicated indelaying and/or reducing the severity of Alzheimer's Disease (Jordan1998).

Failure to produce estrogen has profound physiological consequences infemales. Failure to produce estrogen resulting from incomplete or absentovary development (Turner's Syndrome) causes deficiencies in the skin,bone (e.g., severe osteoporosis), and other organs severely affectingthe life of the afflicted individual (Dodge 1995). In normal women,estrogen production falls sharply upon the onset of menopause, usuallyat about 50 years of age. The effects of the loss of estrogen productioninclude increased atherosclerotic deposits (leading to greatly increaseincidence of heart disease), decreased bone density (osteoporosis), andfluctuations in body temperature among others (Jordan 1998). Often, theeffects of reduced estrogen production are addressed by hormonereplacement therapy (Dodge 1995; Berkow, Beers et al. 1997; Jordan1998).

However, estrogen also has undesirable effects. In menopausal women,supplementation of estrogen is associated with alleviation of theabove-described unwanted effects. But, administration of estrogen isalso associated with increased risks for breast and endometrial canceras well as blood clots (Jordan 1998). The increased risk of endometrialcancer can be addressed by the administration of progesterone (or itssynthetic analog progestin) to re-initiate menstruation and thereby shedpotentially malignant cells, but many older women find this undesirable(Jordan 1998). Breast cancer, however, is by far the greater risk ofestrogen replacement therapy, affecting one woman in every 15 betweenthe ages of 60 and 79 (Jordan 1998).

Thus, for a long time the treatment options for the serious healthproblems caused by a failure to produce estrogen were limited andentailed severe risks. However, the discovery that some agents acted asestrogen agonists in some tissues (e.g., bone) and as an antagonists inother tissues (e.g., breast) provided hope that more effectivetreatments for estrogen loss could be found (Gradishar and Jordan 1997;Gustafsson 1998; Jordan 1998; MacGregor and Jordan 1998). The best knownof these so-called Selective Estrogen Receptor Modulators (“SERMs”),tamoxifen, has been demonstrated to have therapeutic utility in treatingand preventing breast cancer and lowering LDL concentrations; yet,without significant reduction bone density (Jordan 1998; MacGregor andJordan 1998). However, tamoxifen has been associated with endometrialcancer and venous blood clots (Jordan 1998; MacGregor and Jordan 1998).In addition, tumor resistance to tamoxifen can occur (MacGregor andJordan 1998).

Tamoxifen has been followed recently by newer SERMs, in particularraloxifene, that promise to provide many of tamoxifen's benefits withfewer risks (Howell, Downey et al. 1996; Gradishar and Jordan 1997;Gustafsson 1998; Jordan 1998; Purdie 1999; Sato, Grese et al. 1999).These newer SERMs, including idoxifene (Nuttall, Bradbeer et al. 1998),CP-336,156 (Ke, Paralkar et al. 1998), GW5638 (Willson, Norris et al.1997), LY353581 (Sato, Turner et al. 1998) are part of the second- andthird generation of partial estrogen agonists/antagonists. In addition,a new generation of pure antiestrogens such as RU 58,688 (Van de Velde,Nique et al. 1994) have been reported. A large number of additionalpartial and pure estrogen agonist/antagonist compounds and treatmentmodalities have reported recently (Bryant and Dodge 1995; Bryant andDodge 1995; Cullinan 1995; Dodge 1995; Grese 1995; Labrie and Merand1995; Labrie and Merand 1995; Thompson 1995; Audia and Neubauer 1996;Black, Bryant et al. 1996; Thompson 1996; Cullinan 1997; Wilson 1997;Miller, Collini et al. 1999; Palkowitz 1999; Wilson 1999).

However, no one drug candidate has emerged to fill the needs of womenwho require the benefits of estrogen replacement to live productivelives and/or treatments for estrogen-dependent cancers. The efforts todevelop better partial and pure estrogen agonists and antagonists hasbeen aided by several recent developments, including the discovery thathuman estrogen receptor has at least two isoforms (“ERα” and “ERβ”) andthe crystal structure of ERα that have permitted high-resolutionstructure-acitivty relationship studies (Sadler, Cho et al. 1998).Recently, a study of the application of combinatorial synthetic methodscombined with three-dimensional structure-activity analysis to developSERMs having optimal therapeutic profiles was reported (Fink, Mortensenet al. 1999). That study examined several heterocyclic motifs(imidazoles, thiazoles, pyrazoles, oxazoles, and isoxazoles) andidentified certain pyrazole motifs as being well suited forcombinatorial development of SERMs. The relative binding effectivenessof the pyrazoles viz. the other motifs was based on its ability to carryfour substituents in addition to polarity consideration (see p. 215). Inparticular, the study referred the capacity of the pyrazole motif tocarry four substituents explained the binding effectiveness pyrazolescompared to the poor binding results found for the oxazole, thiazole,and isoxazole motifs.

However, despite these recent advances no drug candidate has emerged tofill the needs of women who require the benefits of estrogen replacementto live productive lives and/or treatments for estrogen-dependentcancers. The present invention addresses these and other needs.

3 SUMMARY OF THE INVENTION

The present invention provides isoxazole estrogen receptor agonist andantagonist compounds in addition to methods and compositions fortreating or preventing estrogen receptor-mediated disorders. Thecompounds described herein have been found to have unexpected andsurprising activity in modulating estrogen receptor activity. Thus, thecompounds of the present invention have utility in preventing ortreating estrogen receptor-mediated disorders such as osteoporosis,breast and endometrial cancers, atherosclerosis, and Alzheimer'sdisease.

In a first aspect, the present invention provides compounds having thestructure shown below:

and its pharmaceutically acceptable salts. X₁ and X₂ are selectedindependently from the group consisting of nitrogen and oxygen such thatif one of X₁ and X₂ is nitrogen, then the other of X₁ and X₂ is oxygento form thereby an isoxazole ring structure. R₁ and R₃ are selectedindependently from the group consisting of optionally substitutedloweralkyl, aryl, heteroaryl, cycloalkyl, cycloheteroalkyl, aralkyl,heteroaralkyl, (cycloalkyl)alkyl, and (cycloheteroalkyl)alkyl. R₂ isselected from the group consisting of hydrogen, halo, cyano, nitro,thio, amino, carboxyl, formyl, and optionally substituted loweralkyl,loweralkylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy,cycloalkylcarbonyloxy, cycloheteroalkylcarbonyloxy, aralkycarbonyloxy,heteroaralkylcarbonyloxy, (cycloalkyl)alkylcarbonyloxy,(cycloheteroalkyl)alkylcarbonyloxy, loweralkylcarbonyl, arylcarbonyl,heteroarylcarbonyl, cycloalkylcarbonyl, cycloheteroalkylcarbonyl,aralkycarbonyl, heteroaralkylcarbonyl, (cycloalkyl)alkylcarbonyl,(cycloheteroalkyl)alkylcarbonyl, loweralkylaminocarbonyl,arylaminocarbonyl, aralkylaminocarbonyl, heteroarylaminocarbonyl,heteroaralkylaminocarbonyl, cycloalkylaminocarbonyl,(cycloalkyl)alkylaminocarbonyl, cycloheteroalkylaminocarbonyl,(cycloheteroalkyl)alkylaminocarbonyl, loweralkylcarbonylamino,arylcarbonylamino, heteroarylcarbonylamino, cycloalkylcarbonylamino,cycloheteroalkylcarbonylamino, aralkylcarbonylamino,heteroaralkylcarbonylamino, (cycloalkyl)alkylcarbonylamino,(cycloheteroalkyl)alkylcarbonylamino, loweralkylamino, arylamino,aralkylamino, heteroarylamino, heteroaralkylamino, loweralkylsulfonyl,arylsulfonyl, heteroarylsulfonyl, cycloalkylsulfonyl,cycloheteroalkylsulfonyl, aralkylsulfonyl, heteroaralkylsulfonyl,(cycloalkyl)alkylsulfonyl, (cycloheteroalkyl)alkylsulfonyl,loweralkylsulfinyl, arylsulfinyl, heteroarylsulfinyl,cycloalkylsulfinyl, cycloheteroalkylsulfinyl, aralkylsulfinyl,heteroaralkylsulfinyl, (cycloalkyl)alkylsulfinyl,(cycloheteroalkyl)alkylsulfinyl, loweralkyloxy, aryloxy, heteroaryloxy,cycloalkyloxy, cycloheteroalkyloxy, aralkyloxy, heteroaralkyloxy,(cycloalkyl)alkyloxy, and (cycloheteroalkyl)alkyloxy, loweralkylthio,arylthio, heteroarylthio, cycloalkylthio, cycloheteroalkylthio,aralkylthio, heteroaralkylthio, (cycloalkyl)alkylthio,(cycloheteroalkyl)alkylthio, loweralkylthiocarbonyl, arylthiocarbonyl,heteroarylthiocarbonyl, cycloalkylthiocarbonyl,cycloheteroalkylthiocarbonyl, aralkythiocarbonyloxythiocarbonyl,heteroaralkylthiocarbonyl, (cycloalkyl)alkylthiocarbonyl,(cycloheteroalkyl)alkylthiocarbonyl, heteroarylcarbonylthio,cycloalkylcarbonylthio, cycloheteroalkylcarbonylthio,aralkycarbonylthiooxycarbonylthio, heteroaralkylcarbonylthio,(cycloalkyl)alkylcarbonylthio, (cycloheteroalkyl)alkylcarbonylthio,loweralkyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,cycloalkyloxycarbonyl, cycloheteroalkyloxycarbonyl, aralkyoxycarbonyl,heteroaralkyloxycarbonyl, (cycloalkyl)alkyloxycarbonyl,(cycloheteroalkyl)alkyloxycarbonyl, iminoloweralkyl, iminocycloalkyl,iminocycloheteroalkyl, iminoaralkyl, iminoheteroaralkyl,(cycloalkyl)iminoalkyl, (cycloheteroalkyl)iminoalkyl,(cycloiminoalkyl)alkyl, (cycloiminoheteroalkyl)alkyl, oximinoloweralkyl,oximinocycloalkyl, oximinocycloheteroalkyl, oximinoaralkyl,oximinoheteroaralkyl, (cycloalkyl)oximinoalkyl,(cyclooximinoalkyl)alkyl, (cyclooximinoheteroalkyl)alkyl, and(cycloheteroalkyl)oximinoalkyl.

In one more specific embodiment of the invention, R₂ is selected fromthe group consisting of hydrogen, halo, and optionally substitutedloweralkyl, haloloweralkyl, aryl, aralkyl, heteroaryl, heteroaralkyl,aryloxyalkyl, arylthioalkyl, arylcarbonyl, heteroarylcarbonyl,loweralkylcarbonyl, aminocarbonyl, arylaminocarbonyl,loweralkylaminocarbonyl, aralkylaminocarbonyl,(heterocycloloweralkyl)alkylaminocarbonyl, heteroarylaminocarbonyl,heteroaralkylaminocarbonyl, (cycloloweralkyl)aminocarbonyl, formyl,amino, loweralkylamino, And alkenyl. More particular embodiments arethose for which R₂ is selected from the group consisting of optionallysubstituted phenylcarbonyl,(heterocycloalkyl)loweralkyloxyphenylcarbonyl, hydroxyphenylcarbonyl,halophenylcarbonyl, phenylloweralkylaminocarbonyl,diloweralkylaminocarbonyl, phenylloweralkylaminocarbonyl,hydroxyphenyllowerlakylaminocarbonyl, cycloalkylaminocarbonyl,loweralkylphenylcarbonyl,haloloweralkylsulfonylloweralkyloxyphenylcarbonyl, andnitrophenylcarbonyl. Examples of R₂ substituents within this embodimenthaving useful properties include, but are not limited to,4-(2-piperidin-1-ylethyloxy)phenylcarbonyl, 4-hydroxyphenylcarbonyl,(phenylmethyl)aminocarbonyl,3-(2-oxopyrrolidin-1-yl)propylaminocarbonyl, di-n-butylaminocarbonyl,(4-hydroxyphenylmethyl)aminocarbonyl, (pyridin-3-ylmethyl)aminocarbonyl,(pyridin-2-ylmethyl)aminocarbonyl, dimethylaminocarbonyl,ethylaminocarbonyl, 4-(2-morpholinoethyloxy)phenylcarbonyl,4-(3-dimethylaminopropyloxy)phenylcarbonyl, cyclopropylaminocarbonyl,cyclobutylaminocarbonyl, 4-(2-dimethylaminoethyloxy)phenylcarbonyl,4-[2-(benzylmethylamino)ethyloxy]phenylcarbonyl,4-(1-methylpiperidin-3-ylmethyloxy)phenylcarbonyl,4-[2-(1-methylpyrrolidin-2-yl)ethyloxy]phenylcarbonyl,4-[2-(4-methylpiperazin-1-yl)ethyloxy]phenylcarbonyl,4-(1-methylpiperidin-4-ylmethyloxy)phenylcarbonyl,2-chlorophenylcarbonyl, 3-chlorophenylcarbonyl, 4-chlorophenylcarbonyl,3-nitrophenylcarbonyl, 4-nitrophenylcarbonyl,3,4-dichlorophenylcarbonyl, 4-n-butylphenylcarbonyl,3-hydroxyphenylcarbonyl, 2-hydroxyphenylcarbonyl,4-methoxyphenylcarbonyl, 3-(2-piperidin-1-ylethyloxy)phenylcarbonyl,3-(2-diethylaminoethyloxy)phenylcarbonyl,3-[2-(pyrrolidin-1-yl)ethyloxy]phenylcarbonyl,3-(1-methylpiperidin-3-ylmethyloxy)phenylcarbonyl, and3-(2-dimethylaminoethyloxy)phenylcarbonyl.

In other embodiments of the invention, R₂ is selected from the groupconsisting of optionally substituted phenylcarbonyl,(heterocycloalkyl)loweralkyloxyphenylcarbonyl, hydroxyphenylcarbonyl,halophenylcarbonyl, phenylloweralkylaminocarbonyl,diloweralkylaminocarbonyl, phenylloweralkylaminocarbonyl,hydroxyphenyllowerlakylaminocarbonyl, cycloalkylaminocarbonyl,loweralkylphenylcarbonyl,haloloweralkylsulfonylloweralkyloxyphenylcarbonyl, andnitrophenylcarbonyl, R₁ and R₃ are selected independently from the groupconsisting of optionally substituted aryl and aralkyl, at least one ofR₁ and R₃ is selected independently from the group consisting of phenyl,phenyloxyloweralkyl, and phenylloweralkyl, at least one of R₁ and R₃ issubstituted with at least one hydroxyl or thio group, and at least oneof R₁ and R₃ is substituted optionally with a substituent selected fromthe group consisting of halogen, loweralkyl, halolowerlalkyl,loweralkyloxy, halolowerlakyloxy, carboxy, loweralkyloxycarbonyl,aryloxycarbonyl, (cycloloweralkyl)oxycarbonyl, aralkyloxycarbonyl,heteroaryloxycarbonyl, heteroaralkyloxycarbonyl,(heterocycloloweralkyl)oxycarbonyl, loweralkylsulfinyl,loweralkylsulfonyl, loweralkylthio, arylthio, loweralkylcarbonyloxy,arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy,heteroaralkylcarbonyloxy, (cycloloweralkyl)carbonyloxy,(heterocycloloweralkyl)carbonyloxy, aminocarbonyl,loweraklylaminocarbonyl, arylaminocarbonyl, aralkylaminocarbonyl,heteroarylaminocarbonyl, and heteroaralkylaminocarbonyl. Examples ofspecific useful groups for R₁ and R₃ include without limitation2-methyl-4-hydroxyphenyl, 2-aminocarbonyl-4-hydroxyphenyl,4-methylsulfonylaminophenyl, 3-aminocarbonyl-4-hydroxyphenyl,3-aminocarbonyl-4-methoxyphenyl, 3-chloro-4-hydroxyphenyl,4-methylcarbonyloxyphenyl, 3-n-hexyl-4-hydroxyphenyl,4-n-propylcarbonyloxyphenyl, 3-ethyl-4-hydroxyphenyl,2-methylsulfinyl-4-hydroxyphenyl, 2-ethyl-4-hydroxyphenyl,2-carboxy-4-hydroxyphenyl, 3-fluoro-4-hydroxyphenyl,2-iodo-4-hydroxyphenyl, 2-n-butyl-4-hydroxyphenyl,2-trifluoromethoxyphenyl, and 4-fluorophenyl.

In another aspect, the present invention provides estrogenreceptor-modulating compounds having the following structure:

and their pharmaceutically acceptable salts. X₃ and X₄ are selectedindependently from the group consisting of nitrogen and oxygen such thatif one of X₃ and X₄ is nitrogen, then the other of X₃ and X₄ is oxygento form thereby an isoxazole ring structure. X₅ is —(X₁₀)_(n)—, whereinn is an integer between 1 and 3 and X₁₀, for each value of n, isselected independently from the group consisting of oxygen, —SO_(x)—where xi is and integer between 0 and 2, nitrogen, nitrogen substitutedwith optionally substituted loweralkyl, aryl, aralkyl, heteroaryl,heteroaralkyl, arylcarbonyl, alkylcarbonyl, aralkylcarbonyl,heteroarylcarbonyl, heteroaralkylcarbonyl, and methylene or methine,each optionally substituted from the group consisting of halo, cyano,nitro, thio, amino, carboxyl, formyl, and optionally substitutedloweralkyl, loweralkylcarbonyloxy, arylcarbonyloxy,heteroarylcarbonyloxy, cycloalkylcarbonyloxy,cycloheteroalkylcarbonyloxy, aralkycarbonyloxy,heteroaralkylcarbonyloxy, (cycloalkyl)alkylcarbonyloxy,(cycloheteroalkyl)alkylcarbonyloxy, loweralkylcarbonyl, arylcarbonyl,heteroarylcarbonyl, cycloalkylcarbonyl, cycloheteroalkylcarbonyl,aralkycarbonyl, heteroaralkylcarbonyl, (cycloalkyl)alkylcarbonyl,(cycloheteroalkyl)alkylcarbonyl, loweralkylaminocarbonyl,arylaminocarbonyl, aralkylaminocarbonyl, heteroarylaminocarbonyl,heteroaralkylaminocarbonyl, loweralkylcarbonylamino, arylcarbonylamino,heteroarylcarbonylamino, cycloalkylcarbonylamino,cycloheteroalkylcarbonylamino, aralkylcarbonylamino,heteroaralkylcarbonylamino, (cycloalkyl)alkylcarbonylamino,(cycloheteroalkyl)alkylcarbonylamino, loweralkylamino, arylamino,aralkylamino, heteroarylamino, heteroaralkylamino, loweralkylsulfonyl,arylsulfonyl, heteroarylsulfonyl, cycloalkylsulfonyl,cycloheteroalkylsulfonyl, aralkylsulfonyl, heteroaralkylsulfonyl,(cycloalkyl)alkylsulfonyl, (cycloheteroalkyl)alkylsulfonyl,loweralkylsulfinyl, arylsulfinyl, heteroarylsulfinyl,cycloalkylsulfinyl, cycloheteroalkylsulfinyl, aralkylsulfinyl,heteroaralkylsulfinyl, (cycloalkyl)alkylsulfinyl,(cycloheteroalkyl)alkylsulfinyl, loweralkyloxy, aryloxy, heteroaryloxy,cycloalkyloxy, cycloheteroalkyloxy, aralkyloxy, heteroaralkyloxy,(cycloalkyl)alkyloxy, and (cycloheteroalkyl)alkyloxy, loweralkylthio,arylthio, heteroarylthio, cycloalkylthio, cycloheteroalkylthio,aralkylthio, heteroaralkylthio, (cycloalkyl)alkylthio,(cycloheteroalkyl)alkylthio, loweralkylthiocarbonyl, arylthiocarbonyl,heteroarylthiocarbonyl, cycloalkylthiocarbonyl,cycloheteroalkylthiocarbonyl, aralkythiocarbonyloxlthiocarbonyl,heteroaralkylthiocarbonyl, (cycloalkyl)alkylthiocarbonyl,(cycloheteroalkyl)alkylthiocarbonyl, heteroarylcarbonylthio,cycloalkylcarbonylthio, cycloheteroalkylcarbonylthio,aralkycarbonylthiooxycarbonylthio, heteroaralkylcarbonylthio,(cycloalkyl)alkylcarbonylthio, (cycloheteroalkyl)alkylcarbonylthio,loweralkyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,cycloalkyloxycarbonyl, cycloheteroalkyloxycarbonyl,aralkyoxycarbonyloxloxycarbonyl, heteroaralkyloxycarbonyl,(cycloalkyl)alkyloxycarbonyl, (cycloheteroalkyl)alkyloxycarbonyl,iminoloweralkyl, iminocycloalkyl, iminocycloheteroalkyl, iminoaralkyl,iminoheteroaralkyl, (cycloalkyl)iminoalkyl, and(cycloheteroalkyl)iminoalkyl. X₆-X₉ are selected independently from thegroup consisting of oxygen, sulfur, sulfinyl, nitrogen, and optionallysubstituted methine, and R₄ is selected from the group consisting ofoptionally substituted loweralkyl, aryl, heteroaryl, cycloalkyl,cycloheteroalkyl, aralkyl, heteroaralkyl, (cycloalkyl)alkyl, and(cycloheteroalkyl)alkyl.

In some more specific embodiments of this aspect of the invention, n is1 and X₁₀ is selected from the group consisting of nitrogen, optionallysubstituted nitrogen, and optionally substituted methylene or methine,and R₄ is selected from the group consisting of optionally substitutedaryl, heteroaryl, aralkyl, and heteroaralkyl. Still more specificembodiments include those for which n and X₁₀ have the values justdefined and R₄ is optionally substituted aryl or aralkyl. In still morespecific embodiments, which n, X₁₀ and R₄ have the values just definedR₄ includes at least one hydroxyl, thio, or optionally substitutedloweralkyloxy, aryloxy, heteroaryloxy, loweralkylthio, arylthio,heteroarylthio, loweralkylcarbonyl, arylcarbonyl, or heteroarylcarbonylmoiety. Yet more specific embodiments are those for which n and X₁₀ havethe values just defined wherein R₄ is selected from the group consistingof phenyl, phenyloxyloweralkyl, and phenylloweralkyl, and R₄ includes atleast one hydroxyl, thio, or optionally substituted loweralkyloxy,aryloxy, heteroaryloxy, loweralkylthio, arylthio, heteroarylthio,loweralkylcarbonyl, arylcarbonyl, or heteroarylcarbonyl moiety. Otherembodiments are those for which n and X₁₀ have the values just definedR₄ is selected from the group consisting of phenyl, phenyloxyloweralkyl,and phenylloweralkyl, R₄ includes at least one hydroxyl, thio, oroptionally substituted loweralkyloxy, aryloxy, heteroaryloxy,loweralkylthio, arylthio, heteroarylthio, loweralkylcarbonyl,arylcarbonyl, or heteroarylcarbonyl moiety, and R₄ is furthersubstituted optionally with a moiety selected from the group consistingof halogen, loweralkyl, haloloweralkyl, loweralkyloxy,halolowerlakyloxy, carboxy, loweralkyloxycarbonyl, aryloxycarbonyl,(cycloloweralkyl)oxycarbonyl, aralkyloxycarbonyl, heteroaryloxycarbonyl,heteroaralkyloxycarbonyl, (heterocycloloweralkyl)oxycarbonyl,loweralkylsulfinyl, loweralkylsulfonyl, loweralkylthio, arylthio,loweralkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy,heteroarylcarbonyloxy, heteroaralkylcarbonyloxy,(cycloloweralkyl)carbonyloxy, (heterocycloloweralkyl)carbonyloxy,aminocarbonyl, loweraklylaminocarbonyl, arylaminocarbonyl,aralkylaminocarbonyl, heteroarylaminocarbonyl, cyano, nitro, amino,loweralkylamino, and heteroaralkylaminocarbonyl.

Other embodiments of the present invention include those structures forwhich n is 2 and each X₁₀ is selected independently from the groupconsisting of nitrogen, optionally substituted nitrogen, optionallysubstituted methylene, and optionally substituted methine. In someembodiments, X₁₀ and n have the values just described and R₄ is selectedfrom the group consisting of optionally substituted aryl, heteroaryl,aralkyl, and heteroaralkyl. Still more specific embodiments includethose for which n and X₁₀ have the values just defined and R₄ isoptionally substituted aryl or aralkyl. In still more specificembodiments, which n and X₁₀ have the values just defined, R₄ isoptionally substituted aryl or aralkyl, and R₄ includes at least onehydroxyl, thio, or optionally substituted loweralkyloxy, aryloxy,heteroaryloxy, loweralkylthio. arylthio, heteroarylthio,loweralkylcarbonyl, arylcarbonyl, or heteroarylcarbonyl moiety. Yet morespecific embodiments are those for which n and X₁₀ have the values justdefined wherein R₄ is selected from the group consisting of phenyl,phenyloxyloweralkyl, and phenylloweralkyl, and R₄ includes at least onehydroxyl, thio, or optionally substituted loweralkyloxy, aryloxy,heteroaryloxy, loweralkylthio, arylthio, heteroarylthio,loweralkylcarbonyl, arylcarbonyl, or heteroarylcarbonyl moiety. Otherembodiments are those for which n and X₁₀ have the values just definedwherein R₄ is selected from the group consisting of phenyl,phenyloxyloweralkyl, and phenylloweralkyl, R₄ includes at least onehydroxyl, thio, or optionally substituted loweralkyloxy, aryloxy,heteroaryloxy, loweralkylthio, arylthio, heteroarylthio,loweralkylcarbonyl, arylcarbonyl, or heteroarylcarbonyl moiety, and R₄is further substituted optionally with a moiety selected from the groupconsisting of halogen, loweralkyl, haloloweralkyl, loweralkyloxy,halolowerlakyloxy, carboxy, loweralkyloxycarbonyl, aryloxycarbonyl,(cycloloweralkyl)oxycarbonyl, aralkyloxycarbonyl, heteroaryloxycarbonyl,heteroaralkyloxycarbonyl, (heterocycloloweralkyl)oxycarbonyl,loweralkylsulfinyl, loweralkylsulfonyl, loweralkylthio, arylthio,loweralkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy,heteroarylcarbonyloxy, heteroaralkylcarbonyloxy,(cycloloweralkyl)carbonyloxy, (heterocycloloweralkyl)carbonyloxy,aminocarbonyl, loweraklylaminocarbonyl, arylaminocarbonyl,aralkylaminocarbonyl, heteroarylaminocarbonyl, cyano, nitro, amino,loweralkylamino, and heteroaralkylaminocarbonyl.

Other embodiments of the present invention include compounds for whichX₆-X₉ are selected independently from the group consisting of nitrogenand optionally substituted methine. More particular embodiments arethose for which X₆-X₉ are selected independently from the groupconsisting of nitrogen and optionally substituted methine, and at leastone of X₆-X₉ is methine substituted with a moiety selected from thegroup consisting of loweralkyloxy, aryloxy, heteroaryloxy,loweralkylthio, arylthio, heteroarylthio, loweralkylcarbonyl,arylcarbonyl, and heteroarylcarbonyl. Still more particular embodimentshaving the structural pattern just described include those in which X₇is methine substituted with hydroxy or loweralkyloxy.

In yet another aspect, the present invention provides the presentinvention provides methods for treating or preventing an estrogenreceptor-mediated disorder in a human or animal subject in which anamount of an estrogen receptor-modulating compound of the invention thatis effective to modulate estrogen receptor activity in the subject.Other embodiments provided methods for treating a cell or a estrogenreceptor-mediated disorder in a human or animal subject, comprisingadministering to the cell or to the human or animal subject an amount ofa compound or composition of the invention effective to modulateestrogen receptor activity in the cell or subject. Representativeestrogen receptor-mediated disorders include, for example, osteoporosis,atheroschlerosis, estrogen-mediated cancers (e.g., breast andendometrial cancer), and Alzheimer's disease.

These and other aspects and advantages will become apparent when theDescription below is read in conjunction with the accompanying Examples.

4 DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION

4.1 Definitions

4.1.1 Estrogen Receptor

“Estrogen Receptor” as defined herein refers to any protein in thenuclear receptor gene family that binds estrogen, including, but notlimited to, any isoforms or deletion mutations having thecharacteristics just described. More particularly, the present inventionrelates to estrogen receptor(s) for human and non-human mammals (e.g.,animals of veterinary interest such as horses, cows, sheep, and pigs, aswell as household pets such as cats and dogs). Human estrogen receptorsincluded in the present invention include the α- and β-isoforms(referred to herein as “ERα” and “ERβ”) in addition to any additionalisoforms as recognized by those of skill in the biochemistry arts.

4.1.2 Estrogen Receptor Modulator “Estrogen Receptor Modulator” referherein to a compound that can act as an estrogen receptor agonist orantagonist of estrogen receptor having an IC₅₀ or EC₅₀ with respect toERα and/or ERβ of no more than about 10 μM as determined using the ERαand/or ERβ transactivation assay described hereinbelow (Section5.2.2.3). More typically, estrogen receptor modulators of the inventionhave IC₅₀ or EC₅₀ values (as agonists or antagonists) of not more thanabout 5 μM. Representative compounds of the present invention have beendiscovered to exhibit agonist or antagonist activity viz. estrogenreceptor. Compounds of the present invention preferably exhibit anantagonist or agonist IC₅₀ or EC₅₀ with respect to ERα and/or ERβ of nomore than about 5 μM, more preferably, no more than about 500 nM, evenmore preferably not more than about 1 nM, and most preferably, not morethan about 500 pM, as measured in the ERα and/or ERβ transactivationassays. “IC₅₀” is that concentration of inhibitor which reduces theactivity of a target (e.g., ERα or ERβ) to half-maximal level. “EC₅₀” isthat concentration of modulator that produces half-maximal effect.

4.1.3 Selective Estrogen Receptor Modulator

A “Selective Estrogen Receptor Modulator” (or “SERM”) is a compound thatexhibits activity as an agonist or antagonist of an estrogen receptor(e.g., ERα or ERβ) in a tissue-dependent manner. Thus, as will beapparent to those of skill in the biochemistry and endocrinology arts,compounds of the invention that function as SERMs can act as estrogenreceptor agonists in some tissues (e.g., bone, brain, and/or heart) andas antagonists in other tissue types, such as the breast and/or uterinelining.

4.1.4 Optionally Substituted

“Optionally substituted” refers to the replacement of hydrogen with amonovalent or divalent radical. Suitable substitution groups include,for example, hydroxyl, nitro, amino, imino, cyano, halo, thio,thioamido, amidino, oxo, oxamidino, methoxamidino, imidino, guanidino,sulfonamido, carboxyl, formyl, loweralkyl, haloloweralkyl, loweralkoxy,haloloweralkoxy, loweralkoxyalkyl, alkylcarbonyl, arylcarbonyl,aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, alkylthio,aminoalkyl, cyanoalkyl, and the like. The substitution group can itselfbe substituted. The group substituted onto the substitution group canbe, for example, carboxyl, halo; nitro, amino, cyano, hydroxyl,loweralkyl, loweralkoxy, aminocarbonyl, —SR, thioamido, —SO₃H, —SO₂R orcycloalkyl, where R is typically hydrogen, hydroxyl or loweralkyl. Whenthe substituted substituent includes a straight chain group, thesubstitution can occur either within the chain (e.g., 2-hydroxypropyl,2-aminobutyl, and the like) or at the chain terminus (e.g.,2-hydroxyethyl, 3-cyanopropyl, and the like). Substituted substitutentscan be straight chain, branched or cyclic arrangements of covalentlybonded carbon or heteroatoms.

4.1.5 Loweralkyl and Related Terms “Lower alkyl” as used herein refersto branched or straight chain alkyl groups comprising one to ten carbonatoms that independently are unsubstituted or substituted, e.g., withone or more halogen, hydroxyl or other groups. Examples of loweralkylgroups include, but are not limited to, methyl, ethyl, propyl,isopropyl, n-butyl, t-butyl, n-hexyl, neopentyl, trifluoromethyl,pentafluoroethyl, and the like.

“Alkylenyl” refers to a divalent straight chain or branched chainsaturated aliphatic radical having from 1 to 20 carbon atoms. Typicalalkylenyl groups employed in compounds of the present invention areloweralkylenyl groups that have from 1 to about 6 carbon atoms in theirbackbone. “Alkenyl” refers herein to straight chain, branched, or cyclicradicals having one or more double bonds and from 2 to 20 carbon atoms.“Alkynyl” refers herein to straight chain, branched, or cyclic radicalshaving one or more triple bonds and from 2 to 20 carbon atoms.

The term “haloloweralkyl” refers to a loweralkyl radical substitutedwith one or more halogen atoms.

“Loweralkoxy” as used herein refers to RO— wherein R is loweralkyl.Representative examples of loweralkoxy groups include methoxy, ethoxy,t-butoxy, trifluoromethoxy and the like.

“Loweralkylthio” as used herein refers to RS— wherein R is loweralkyl.

The term “alkoxyalkyl” refers to the group -alk₁-O-alk₂ where alk₁ isalkylenyl or alkenyl, and alk₂ is alkyl or alkenyl. The term“loweralkoxyalkyl” refers to an alkoxyalkyl where alk₁ is loweralkylenylor loweralkenyl, and alk₂ is loweralkyl or loweralkenyl. The term“aryloxyalkyl” refers to the group -alkylenyl-O-aryl. The term“aralkoxyalkyl” refers to the group -alkylenyl-O-aralkyl, where aralkylis a loweraralkyl.

“Cycloalkyl” refers to a mono- or polycyclic, loweralkyl substituent.Typical cycloalkyl substituents have from 3 to 8 backbone (i.e., ring)atoms in which each backbone atom is optionally substituted carbon. Whenused in context with cycloalkyl substituents, the term “polycyclic”refers herein to fused, non-fused cyclic carbon structures andspirocycles. Examples of cycloalkyl groups include, but are not limitedto, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl, bornyl,norbornyl, and the like.

The term “cycloheteroalkyl” refers herein to cycloalkyl substituentsthat have from 1 to 5, and more typically from 1 to 4 heteroatoms (i.e.,non-carbon atoms such as nitrogen, sulfur, and oxygen) in the ringstructure, with the balance of atoms in the ring being optionallysubstituted carbon. Representative heterocycloalkyl moieties include,for example, morpholino, piperazinyl, piperidinyl, pyrrolidinyl,methylpryolidinyl, pyrrolidinone-yl, and the like.

The terms “(cycloalkyl)alkyl” and “(cycloheteroalkyl)alkyl” refer toalkyl chains substituted with cycloalkyl and cycloheteroalkyl groupsrespectively.

The term “haloalkoxy” refers to an alkoxy radical substituted with oneor more halogen atoms. The term “haloloweralkoxy” refers to aloweralkoxy radical substituted with one or more halogen atoms.

4.1.6 Halo

“Halo” refers herein to a halogen radical, such as fluorine, chlorine,bromine, or iodine.

4.1.7 Aryl and Related Terms

“Aryl” refers to monocyclic and polycyclic aromatic groups, or fusedring systems having at least one aromatic ring, having from 3 to 14backbone carbon atoms. Examples of aryl groups include withoutlimitation phenyl, naphthyl, dihydronaphtyl, tetrahydronaphthyl, and thelike.

“Aralkyl” refers to an alkyl group substituted with an aryl group.Typically, aralkyl groups employed in compounds of the present inventionhave from 1 to 6 carbon atoms incorporated within the alkyl portion ofthe aralkyl group. Suitable aralkyl groups employed in compounds of thepresent invention include, for example, benzyl, picolyl, and the like.

4.1.8 Heteroaryl and Related Terms

The term “heteroaryl” refers herein to aryl groups having from one tofour heteroatoms as ring atoms in an aromatic ring with the remainder ofthe ring atoms being aromatic or non-aromatic carbon atoms. When used inconnection with aryl substituents, the term “polycyclic” refers hereinto fused and non-fused cyclic structures in which at least one cyclicstructure is aromatic, such as, for example, benzodioxozolo, naphthyl,and the like. Exemplary heteroaryl moieties employed as substituents incompounds of the present invention include pyridyl, pyrimidinyl,thiazolyl, indolyl, imidazolyl, oxadiazolyl, tetrazolyl, pyrazinyl,triazolyl, thiophenyl, furanyl, quinolinyl, purinyl, benzothiazolyl,benzopyridyl, and benzimidazolyl, and the like.

4.1.9 Amino and Related Terms

“Amino” refers herein to the group —NH₂. The term “loweralkylamino”refers herein to the group —NRR′ where R and R′ are each independentlyselected from hydrogen or loweralkyl. The term “arylamino” refers hereinto the group —NRR′ where R is aryl and R′ is hydrogen, loweralkyl, aryl,or aralkyl. The term “aralkylamino” refers herein to the group —NRR′where R is aralkyl and R′ is hydrogen, loweralkyl, aryl, or aralkyl. Theterms “heteroarylamino” and heteroaralkylamino” are defined by analogyto arylamino and aralkylamino.

The term “aminocarbonyl” refers herein to the group —C(O)—NH₂. The terms“loweralkylaminocarbonyl”, arylaminocarbonyl”, “aralkylaminocarbonyl”,“heteroarylaminocarbonyl”, and “heteroaralkylaminocarbonyl” refer to—C(O)NRR′ where R and R′ independently are hydrogen and optionallysubstituted loweralkyl, aryl, aralkyl, heteroaryl, and heteroaralkylrespectively by analogy to the corresponding terms above.

4.1.10 Thio, Sulfonyl, Sulfinyl and Related Terms

The term “thio” refers to —SH. The terms “loweralkylthio”, “arylthio”,“heteroarylthio”, “cycloalkylthio”, “cycloheteroalkylthio”,“aralkylthio”, “heteroaralkylthio”, “(cycloalkyl)alkylthio”, and“(cycloheteroalkyl)alkylthio” refer to —SR, where R is optionallysubstituted loweralkyl, aryl, heteroaryl, cycloalkyl, cycloheteroalkyl,aralkyl, heteroaralkyl, (cycloalkyl)alkyl, and (cycloheteroalkyl)alkylrespectively.

The term “sulfonyl” refers herein to the group —SO₂—. The terms“loweralkylsulfonyl”, “arylsulfonyl”, “heteroarylsulfonyl”,“cycloalkylsulfonyl”, “cycloheteroalkylsulfonyl”, “aralkylsulfonyl”,“heteroaralkylsulfonyl”, “(cycloalkyl)alkylsulfonyl”, and“(cycloheteroalkyl)alkylsulfonyl” refer to —SO₂R where R is optionallysubstituted loweralkyl, aryl, heteroaryl, cycloalkyl, cycloheteroalkyl,aralkyl, heteroaralkyl, (cycloalkyl)alkyl, and (cycloheteroalkyl)alkylrespectively.

The term “sulfinyl” refers herein to the group —SO—. The terms“loweralkylsulfinyl”, “arylsulfinyl”, “heteroarylsulfinyl”,“cycloalkylsulfinyl”, “cycloheteroalkylsulfinyl”, “aralkylsulfinyl”,“heteroaralkylsulfinyl”, “(cycloalkyl)alkylsulfinyl”, and“(cycloheteroalkyl)alkylsulfinyl” refer to —SOR where R is optionallysubstituted loweralkyl, aryl, heteroaryl, cycloalkyl, cycloheteroalkyl,aralkyl, heteroaralkyl, (cycloalkyl)alkyl, and (cycloheteroalkyl)alkylrespectively.

4.1.11 Formyl, Carboxyl, Carbonyl, Thiocarbonyl, and Related Terms

“Formyl” refers to —C(O)H.

“Carboxyl” refers to —C(O)OH.

“Carbonyl” refers to the divalent group —C(O)—. The terms“loweralkylcarbonyl”, “arylcarbonyl”, “heteroarylcarbonyl”,“cycloalkylcarbonyl”, “cycloheteroalkylcarbonyl”, “aralkycarbonyl”,“heteroaralkylcarbonyl”, “(cycloalkyl)alkylcarbonyl”, and“(cycloheteroalkyl)alkylcarbonyl” refer to —C(O)R, where R is optionallysubstituted loweralkyl, aryl, heteroaryl, cycloalkyl, cycloheteroalkyl,aralkyl, heteroaralkyl, (cycloalkyl)alkyl, and (cycloheteroalkyl)alkylrespectively.

“Thiocarbonyl” refers to the group —C(S)—. The terms“loweralkylthiocarbonyl”, “arylthiocarbonyl”, “heteroarylthiocarbonyl”,“cycloalkylthiocarbonyl”, “cycloheteroalkylthiocarbonyl”,“aralkythiocarbonyloxlthiocarbonyl”, “heteroaralkylthiocarbonyl”,“(cycloalkyl)alkylthiocarbonyl”, and“(cycloheteroalkyl)alkylthiocarbonyl” refer to —C(S)R, where R isoptionally substituted loweralkyl, aryl, heteroaryl, cycloalkyl,cycloheteroalkyl, aralkyl, heteroaralkyl, (cycloalkyl)alkyl, and(cycloheteroalkyl)alkyl respectively.

“Carbonyloxy” refers generally to the group —C(O)—O—. The terms“loweralkylcarbonyloxy”, “arylcarbonyloxy”, “heteroarylcarbonyloxy”,“cycloalkylcarbonyloxy”, “cycloheteroalkylcarbonyloxy”,“aralkycarbonyloxy”, “heteroaralkylcarbonyloxy”,“(cycloalkyl)alkylcarbonyloxy”, “(cycloheteroalkyl)alkylcarbonyloxy”refer to —C(O)OR, where R is optionally substituted loweralkyl, aryl,heteroaryl, cycloalkyl, cycloheteroalkyl, aralkyl, heteroaralkyl,(cycloalkyl)alkyl, and (cycloheteroalkyl)alkyl respectively.

“Oxycarbonyl” refers to the group —O—C(O)—. The terms“loweralkyloxycarbonyl”, “aryloxycarbonyl”, “heteroaryloxycarbonyl”,“cycloalkyloxycarbonyl”, “cycloheteroalkyloxycarbonyl”,“aralkyoxycarbonyloxloxycarbonyl”, “heteroaralkyloxycarbonyl”,“(cycloalkyl)alkyloxycarbonyl”, “(cycloheteroalkyl)alkyloxycarbonyl”refer to —O—C(O)R, where R is optionally substituted loweralkyl, aryl,heteroaryl, cycloalkyl, cycloheteroalkyl, aralkyl, heteroaralkyl,(cycloalkyl)alkyl, and (cycloheteroalkyl)alkyl respectively.

“Carbonylamino” refers to the group —NH—C(O)—. The terms“loweralkylcarbonylamino”, “arylcarbonylamino”,“heteroarylcarbonylamino”, “cycloalkylcarbonylamino”,“cycloheteroalkylcarbonylamino”, “aralkylcarbonylamino”,“heteroaralkylcarbonylamino”, “(cycloalkyl)alkylcarbonylamino”, and“(cycloheteroalkyl)alkylcarbonylamino” refer to —NH—C(O)R, where R isoptionally substituted loweralkyl, aryl, heteroaryl, cycloalkyl,cycloheteroalkyl, aralkyl, heteroaralkyl, (cycloalkyl)alkyl, or(cycloheteroalkyl)alkyl respectively. In addition, the present inventionincludes N-substituted carbonylamino (—NR′C(O)R), where R′ is optionallysubstituted loweralkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl andR retains the previous defintion.

“Carbonylthio” refers to the group —C(O)—S—. The terms“loweralkylcarbonylthio”, “arylcarbonylthio”, “heteroarylcarbonylthio”,“cycloalkylcarbonylthio”, “cycloheteroalkylcarbonylthio”,“aralkycarbonylthio”, “heteroaralkylcarbonylthio”,“(cycloalkyl)alkylcarbonylthio”, “(cycloheteroalkyl)alkylcarbonylthio”refer to —C(O)SR, where R is optionally substituted loweralkyl, aryl,heteroaryl, cycloalkyl, cycloheteroalkyl, aralkyl, heteroaralkyl,(cycloalkyl)alkyl, and (cycloheteroalkyl)alkyl respectively.

4.1.12 Guanidino or Guanidyl

As used herein, the term “guanidino” or “guanidyl” refers to moietiesderived from guanidine, H₂N—C(═NH)—NH₂. Such moieties include thosebonded at the nitrogen atom carrying the formal double bond (the“2”-position of the guanidine, e.g., diaminomethyleneamino, (H₂N)₂C═NH—)and those bonded at either of the nitrogen atoms carrying a formalsingle bond (the “1-” and/or “3”-positions of the guanidine, e.g.,H₂N—C(═NH)—NH—). The hydrogen atoms at either nitrogen can be replacedwith a suitable substituent, such as loweralkyl, aryl, or loweraralkyl.

4.1.13 Amidino

As used herein, the term “amidino” refers to the moieties R—C(═N)—NR′—(the radical being at the “N¹” nitrogen) and R(NR′)C═N— (the radicalbeing at the “N²” nitrogen), where R and R′ can be hydrogen, loweralkyl,aryl, or loweraralkyl.

4.1.14 Imino and Oximino

The term “imino” refers to the group —C(═NR)—, where R can be hydrogenor optionally substituted loweralkyl, aryl, heteroaryl, or heteroaralkylrespectively. The terms “iminoloweralkyl”, “iminocycloalkyl”,“iminocycloheteroalkyl”, “iminoaralkyl”, “iminoheteroaralkyl”,“(cycloalkyl)iminoalkyl”, “(cycloiminoalkyl)alkyl”,“(cycloiminoheteroalkyl)alkyl”, and “(cycloheteroalkyl)iminoalkyl” referto optionally substituted loweralkyl, cycloalkyl, cycloheteroalkyl,aralkyl, heteroaralkyl, (cycloalkyl)alkyl, and (cycloheteroalkyl)alkylgroups that include an imino group, respectively.

The term “oximino” refers to the group —C(═NOR)—, where R can behydrogen (“hydroximino”) or optionally substituted loweralkyl, aryl,heteroaryl, or heteroaralkyl respectively. The terms“oximinoloweralkyl”, “oximinocycloalkyl”, “oximinocycloheteroalkyl”,“oximinoaralkyl”, “oximinoheteroaralkyl”, “(cycloalkyl)oximinoalkyl”,“(cyclooximinoalkyl)alkyl”, “(cyclooximinoheteroalkyl)alkyl”, and(cycloheteroalkyl)oximinoalkyl” refer to optionally substitutedloweralkyl, cycloalkyl, cycloheteroalkyl, aralkyl, heteroaralkyl,(cycloalkyl)alkyl, and (cycloheteroalkyl)alkyl groups that include anoximino group, respectively.

4.1.15 Methylene and Methine

The term “methylene” as used herein refers to an unsubstituted,monosubstituted, or disubstituted carbon atom having a formal sp³hybridization (i.e., —CRR′—, where R and R′ are hydrogen or independentsubstituents).

The term “methine” as used herein refers to an unsubstituted or carbonatom having a formal sp² hybridization (i.e., —CR═ or ═CR—, where R ishydrogen a substituent).

4.2 Compounds of the Invention

The present invention provides compounds that have useful agonist and/orantagonist activity with respect to mammalian estrogen receptors inaddition to compounds, compositions, and methods useful for treatingestrogen receptor-mediated disorders in mammals. More particularly, thecompounds of the present invention have been found to possess asurprising degree of activity with respect to the α- and β-isoforms ofhuman estrogen receptor. Thus, the compounds, compositions, and methodsdescribed herein have utility in preventing and/or treating a widevariety of estrogen receptor-mediated disorders including, but notlimited to, osteoporosis, breast cancer, uterine cancer, and congestiveheart disease.

In a first aspect, the present invention provides compounds having thestructure (Compound 1):

and its pharmaceutically acceptable salts, X₁ and X₂ are selectedindependently from the group consisting of nitrogen and oxygen such thatif one of X₁ and X₂ is nitrogen, then the other of X₁ and X₂ is oxygento form thereby an isoxazole ring structure. Thus, the generic structureshown above encompasses the following regioisomers:

depending on the identities of X₁ and X₂. R₁ and R₃ are selectedindependently from the group consisting of optionally substitutedloweralkyl, aryl, heteroaryl, cycloalkyl, cycloheteroalkyl, aralkyl,heteroaralkyl, (cycloalkyl)alkyl, and (cycloheteroalkyl)alkyl. R₂ isselected from the group consisting of hydrogen, halo, cyano, nitro,thio, amino, carboxyl, formyl, and optionally substituted loweralkyl,loweralkylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy,cycloalkylcarbonyloxy, cycloheteroalkylcarbonyloxy, aralkycarbonyloxy,heteroaralkylcarbonyloxy, (cycloalkyl)alkylcarbonyloxy,(cycloheteroalkyl)alkylcarbonyloxy, loweralkylcarbonyl, arylcarbonyl,heteroarylcarbonyl, cycloalkylcarbonyl, cycloheteroalkylcarbonyl,aralkycarbonyl, heteroaralkylcarbonyl, (cycloalkyl)alkylcarbonyl,(cycloheteroalkyl)alkylcarbonyl, loweralkylaminocarbonyl,arylaminocarbonyl, aralkylaminocarbonyl, heteroarylaminocarbonyl,heteroaralkylaminocarbonyl, cycloalkylaminocarbonyl,(cycloalkyl)alkylaminocarbonyl, cycloheteroalkylaminocarbonyl,(cycloheteroalkyl)alkylaminocarbonyl, loweralkylcarbonylamino,arylcarbonylamino, heteroarylcarbonylamino, cycloalkylcarbonylamino,cycloheteroalkylcarbonylamino, aralkylcarbonylamino,heteroaralkylcarbonylamino, (cycloalkyl)alkylcarbonylamino,(cycloheteroalkyl)alkylcarbonylamino, loweralkylamino, arylamino,aralkylamino, heteroarylamino, heteroaralkylamino, loweralkylsulfonyl,arylsulfonyl, heteroarylsulfonyl, cycloalkylsulfonyl,cycloheteroalkylsulfonyl, aralkylsulfonyl, heteroaralkylsulfonyl,(cycloalkyl)alkylsulfonyl, (cycloheteroalkyl)alkylsulfonyl,loweralkylsulfinyl, arylsulfinyl, heteroarylsulfinyl,cycloalkylsulfinyl, cycloheteroalkylsulfinyl, aralkylsulfinyl,heteroaralkylsulfinyl, (cycloalkyl)alkylsulfinyl,(cycloheteroalkyl)alkylsulfinyl, loweralkyloxy, aryloxy, heteroaryloxy,cycloalkyloxy, cycloheteroalkyloxy, aralkyloxy, heteroaralkyloxy,(cycloalkyl)alkyloxy, and (cycloheteroalkyl)alkyloxy, loweralkylthio,arylthio, heteroarylthio, cycloalkylthio, cycloheteroalkylthio,aralkylthio, heteroaralkylthio, (cycloalkyl)alkylthio,(cycloheteroalkyl)alkylthio, loweralkylthiocarbonyl, arylthiocarbonyl,heteroarylthiocarbonyl, cycloalkylthiocarbonyl,cycloheteroalkylthiocarbonyl, aralkythiocarbonyloxythiocarbonyl,heteroaralkylthiocarbonyl, (cycloalkyl)alkylthiocarbonyl,(cycloheteroalkyl)alkylthiocarbonyl, heteroarylcarbonylthio,cycloalkylcarbonylthio, cycloheteroalkylcarbonylthio,aralkycarbonylthiooxycarbonylthio, heteroaralkylcarbonylthio,(cycloalkyl)alkylcarbonylthio, (cycloheteroalkyl)alkylcarbonylthio,loweralkyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,cycloalkyloxycarbonyl, cycloheteroalkyloxycarbonyl, aralkyoxycarbonyl,heteroaralkyloxycarbonyl, (cycloalkyl)alkyloxycarbonyl,(cycloheteroalkyl)alkyloxycarbonyl, iminoloweralkyl, iminocycloalkyl,iminocycloheteroalkyl, iminoaralkyl, iminoheteroaralkyl,(cycloalkyl)iminoalkyl, (cycloheteroalkyl)iminoalkyl,(cycloiminoalkyl)alkyl, (cycloiminoheteroalkyl)alkyl, oximinoloweralkyl,oximinocycloalkyl, oximinocycloheteroalkyl, oximinoaralkyl,oximinoheteroaralkyl, (cycloalkyl)oximinoalkyl,(cyclooximinoalkyl)alkyl, (cyclooximinoheteroalkyl)alkyl, and(cycloheteroalkyl)oximinoalkyl.

In one embodiment, R₁ and R₃ are selected independently from the groupconsisting of optionally substituted cycloalkyl, cycloheteroalkyl,(cycloalkyl)alkyl, and (cycloheteroalkyl)alkyl. Examples of such groupsinclude without limitation cyclohexyl, piperidinyl, adamantyl, andquinuclidyl, each optionally substituted. Other examples includecyclohexylmethyl, 2-cyclohexylethyl, and adamantylmethyl, again, eachoptionally substituted. In another embodiment, R₁ and R₃ are selectedindependently from the group consisting of optionally substituted aryl,heteroaryl, aralkyl, and heteroaralkyl. More particular embodiments ofthe invention are those for which R₁ and R₃ are selected independentlyfrom the group consisting of optionally substituted heteroaryl andheteroaralkyl, such as pyridinyl, hydroxcypyridyl, methoxypyridyl,pyridylmethyl, and the like.

Alternatively, R₁ and R₃ are selected independently from the groupconsisting of optionally substituted aryl and aralkyl. In more specificembodiments, R₁ and R₃ are selected independently from the groupconsisting of optionally substituted aryl and aralkyl and at least oneof R₁ and R₃ is substituted with at least one hydroxyl, alkyloxy,aryloxy, thio, alkylthio, or arylthio group. Still more specificembodiments are those which R₁ and R₃ are selected independently fromthe group consisting of optionally substituted aryl and aralkyl and atleast one of R₁ and R₃ is substituted with at least one hydroxyl,alkyloxy, aryloxy, thio, alkylthio, or arylthio group are those whereinat least one of R₁ and R₃ is selected independently from the groupconsisting of phenyl, phenyloxyloweralkyl, and phenylloweralkyl andinclude the substitutions just listed. Examples of useful groups includewithout limitation 4-hydoxyphenyl, phenylmethyl, 4-hydroxyphenymethyl,3-hydroxyphenylmethyl, 2-thio-4-hydroxyphenylmethyl,2-(4-hydroxyphenyl)ethyl, phenyloxy)methyl, 4-methoxyphenyl,2-hydroxyphenyl, 3-(phenylthio)-4-hydroxyphenyl, and3-methylphenyl-4-hydroxyphenyl. In a still more specific embodiment, thepresent invention includes compounds of the structure shown for Compound1 for which R₁ and R₃ are selected independently from the groupconsisting of optionally substituted aryl and aralkyl such that at leastone of R₁ and R₃ is substituted with at least one hydroxyl, alkyloxy,aryloxy, thio, alkylthio, or arylthio group and at least one of R₁ andR₃ is selected independently from the group consisting of phenyl,phenyloxyloweralkyl, and phenylloweralkyl, and further wherein at leastone of R₁ and R₃ is substituted optionally with a substituent selectedfrom the group consisting of halogen, nitro, cyano, loweralkyl,halolowerlalkyl, loweralkyloxy, haloloweralkyloxy, carboxy,loweralkyloxycarbonyl, aryloxycarbonyl, (cycloloweralkyl)oxycarbonyl,aralkyloxycarbonyl, heteroaryloxycarbonyl, heteroaralkyloxycarbonyl,(heterocycloloweralkyl)oxycarbonyl, loweralkylsulfinyl,loweralkylsulfonyl, loweralkylthio, arylthio, loweralkylcarbonyloxy,arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy,heteroaralkylcarbonyloxy, (cycloloweralkyl)carbonyloxy,alkylsulfonylamino, (heterocycloloweralkyl)carbonyloxy, aminocarbonyl,lowerakylaminocarbonyl, arylaminocarbonyl, aralkylaminocarbonyl,heteroarylaminocarbonyl, and heteroaralkylaminocarbonyl. Yet morespecific embodiments includes those just recited for which at least oneof R₁ and R₃ is further substituted optionally with a substituentselected from the group consisting of halogen, nitro, cyano, loweralkyl,halolowerlalkyl, loweralkyloxy, halolowerlakyloxy, carboxy,loweralkylthio, aminocarbonyl, and loweralkylsulfinyl. Examples ofspecific useful groups of this embodiment include without limitation2-methyl-4-hydroxyphenyl, 2-aminocarbonyl-4-hydroxyphenyl,4-methylsulfonylaminophenyl, 3-aminocarbonyl-4-hydroxyphenyl,3-aminocarbonyl-4-methoxyphenyl, 3-chloro-4-chloro-4-hydroxyphenyl,4-methylcarbonyloxyphenyl, 3-n-hexyl-4-hydroxyphenyl,4-n-propylcarbonyloxyphenyl, 3-ethyl-4-hydroxyphenyl,2-methylsulfinyl-4-hydroxyphenyl, 2-ethyl-4-hydroxyphenyl,2-carboxy-4-hydroxyphenyl, 3-fluoro-4-hydroxyphenyl,2-iodo-4-hydroxyphenyl, 2-n-butyl-4-hydroxyphenyl,2-trifluoromethoxyphenyl, and 4-fluorophenyl.

In other embodiments of the present invention, Compound 1 above includescompounds in R₂ is selected from the group consisting of hydrogen, halo,and optionally substituted loweralkyl, haloloweralkyl, aryl, aralkyl,heteroaryl, heteroaralkyl, aryloxyalkyl, arylthioalkyl, arylcarbonyl,heteroarylcarbonyl, loweralkylcarbonyl, aminocarbonyl,arylaminocarbonyl, loweralkylaminocarbonyl, aralkylaminocarbonyl,(heterocycloloweralkyl)alkylaminocarbonyl, heteroarylaminocarbonyl,heteroaralkylaminocarbonyl, (cycloloweralkyl)aminocarbonyl, formyl,amino, loweralkylamino, and alkenyl. Particular examples of theseembodiments include those for which R₂ is selected from the groupconsisting of hydrogen and halo. Other particular examples of theseembodiments are those in which R₂ is selected from the group consistingof optionally substituted phenyl, phenylloweralkyl, hydroxyphenyl,loweralkyloxyphenyl, haloloweralkylsulfonylloweralkyloxyphenyl,diloweralkylaminoloweralkyloxyphenyl,(cycloaminoloweralkyl)loweralkyloxyphenyl, and(heterocycloalkyl)loweralkyloxyphenyl. Specific examples of such groupsinclude phenylmethyl, 4-hydroxyphenyl, 2-(piperidin-1-yl)ethyloxyphenyl,4-hydroxyphenyloxymethyl, and 2-(piperidin-1-yl)ethyloxyphenylmethyl.Still other particular embodiments are those for which R₂ is selectedfrom the group consisting of optionally substituted loweralkyl,haloloweralkyl, hydroxyalkyl, phenyloxyloweralkyl,hydroxyphenyloweralkyl, haloloweralkylsulfonylloweralkyl, andphenylthioloweralkyl.

In still other embodiments of Compound 1 above R₂ is selected from thegroup consisting of optionally substituted phenylcarbonyl,(heterocycloalkyl)loweralkyloxyphenylcarbonyl, hydroxyphenylcarbonyl,halophenylcarbonyl, phenylloweralkylaminocarbonyl,diloweralkylaminocarbonyl, phenylloweralkylaminocarbonyl,hydroxyphenyllowerlakylaminocarbonyl, cycloalkylaminocarbonyl,loweralkylphenylcarbonyl,haloloweralkylsulfonylloweralkyloxyphenylcarbonyl, andnitrophenylcarbonyl. Examples of R₂ substituents within this embodimenthaving useful properties include, but are not limited to,4-(2-piperidin-1-ylethyloxy)phenylcarbonyl, 4-hydroxyphenylcarbonyl,(phenylmethyl)aminocarbonyl,3-(2-oxopyrrolidin-1-yl)propylaminocarbonyl, di-n-butylaminocarbonyl,(4-hydroxyphenylmethyl)aminocarbonyl, (pyridin-3-ylmethyl)aminocarbonyl,(pyridin-2-ylmethyl)aminocarbonyl, dimethylaminocarbonyl,ethylaminocarbonyl, 4-(2-morpholinoethyloxy)phenylcarbonyl,4-(3-dimethylaminopropyloxy)phenylcarbonyl, cyclopropylaminocarbonyl,cyclobutylaminocarbonyl, 4-(2-dimethylaminoethyloxy)phenylcarbonyl,4-[2-(benxylmethylamino)ethyloxy]phenylcarbonyl,4-(1-methylpiperidin-3-ylmethyloxy)phenylcarbonyl,4-[2-(1-methylpyrrolidin-2-yl)ethyloxy]phenylcarbonyl,4-[2-(4-methylpiperazin-1-yl)ethyloxy]phenylcarbonyl,4-(1-methylpiperidin-4-ylmethyloxy)phenylcarbonyl,2-chlorophenylcarbonyl, 3-chlorophenylcarbonyl, 4-chlorophenylcarbonyl,3-nitrophenylcarbonyl, 4-nitrophenylcarbonyl,3,4-dichlorophenylcarbonyl, 4-n-butylphenylcarbonyl,3-hydroxyphenylcarbonyl, 2-hydroxyphenylcarbonyl,4-methoxyphenylcarbonyl, 3-(2-piperidin-1-ylethyloxy)phenylcarbonyl,3-(2-diethylaminoethyloxy)phenylcarbonyl,3-[2-(pyrrolidin-1-yl)ethyloxy]phenylcarbonyl,3-(1-methylpiperidin-3-ylmethyloxy)phenylcarbonyl, and3-(2-dimethylaminoethyloxy)phenylcarbonyl.

In other embodiments, the compounds, compositions, and methods providedby the present invention include those compounds having the structure ofCompound 1 above for which R₂ is selected from the group consisting ofoptionally substituted phenylcarbonyl,(heterocycloalkyl)loweralkyloxyphenylcarbonyl, hydroxyphenylcarbonyl,halophenylcarbonyl, phenylloweralkylaminocarbonyl,diloweralkylaminocarbonyl, phenylloweralkylaminocarbonyl,hydroxyphenyllowerlakylaminocarbonyl, cycloalkylaminocarbonyl,loweralkylphenylcarbonyl,haloloweralkylsulfonylloweralkyloxyphenylcarbonyl, andnitrophenylcarbonyl, and R₁ and R₃ are selected independently from thegroup consisting of optionally substituted cycloalkyl, cycloheteroalkyl,(cycloalkyl)alkyl, and (cycloheteroalkyl)alkyl. Still other embodimentsinclude those for which R₂ is selected from the group consisting ofoptionally substituted phenylcarbonyl,(heterocycloalkyl)loweralkyloxyphenylcarbonyl, hydroxyphenylcarbonyl,halophenylcarbonyl, phenylloweralkylaminocarbonyl,diloweralkylaminocarbonyl, phenylloweralkylaminocarbonyl,hydroxyphenyllowerlakylaminocarbonyl, cycloalkylaminocarbonyl,lowerallkylphenylcarbonyl,haloloweralkylsulfonylloweralkyloxyphenylcarbonyl, andnitrophenylcarbonyl, and R₁ and R₃ are selected independently from thegroup consisting of optionally substituted aryl, heteroaryl, aralkyl,and heteroaralkyl. Of the latter embodiments, more specific embodimentsinclude those for which R₁ and R₃ are selected independently from thegroup consisting of optionally substituted aryl and aralkyl, and, stillmore particularly, those compounds wherein R₂ is selected from the groupconsisting of optionally substituted phenylcarbonyl,(heterocycloalkyl)loweralkyloxyphenylcarbonyl, hydroxyphenylcarbonyl,halophenylcarbonyl, phenylloweralkylaminocarbonyl,diloweralkylaminocarbonyl, phenylloweralkylaminocarbonyl,hydroxyphenyllowerlakylaminocarbonyl, cycloalkylaminocarbonyl,loweralkylphenylcarbonyl,haloloweralkylsulfonylloweralkyloxyphenylcarbonyl, andnitrophenylcarbonyl, R₁ and R₃ are selected independently from the groupconsisting of optionally substituted aryl and aralkyl, and at least oneof R₁ and R₃ is selected independently from the group consisting ofphenyl, phenyloxyloweralkyl, and phenylloweralkyl, and, morespecifically those compounds having the just-described substitutionpattern wherein at least one of R₁ and R₃ is substituted with at leastone hydroxyl or thio group. Yet more specific embodiments are those forwhich R₂ is selected from the group consisting of optionally substitutedphenylcarbonyl, (heterocycloalkyl)loweralkyloxyphenylcarbonyl,hydroxyphenylcarbonyl, halophenylcarbonyl,phenylloweralkylaminocarbonyl, diloweralkylaminocarbonyl,phenylloweralkylaminocarbonyl, hydroxyphenyllowerlakylaminocarbonyl,cycloalkylaminocarbonyl, loweralkylphenylcarbonyl,haloloweralkylsulfonylloweralkyloxyphenylcarbonyl, cnanitrophenylcarbonyl, R₁ and R₃ are selected independently from the groupconsisting of optionally substituted aryl and aralkyl, at least one ofR₁ and R₃ is selected independently from the group consisting of phenyl,phenyloxyloweralkyl, and phenylloweralkyl, at least one of R₁ and R₃ issubstituted with at least one hydroxyl or thio group, and at least oneof R₁ and R₃ is substituted optionally with a substituent selected fromthe group consisting of halogen, loweralkyl, halolowerlalkyl,loweralkyloxy, halolowerlakyloxy, carboxy, loweralkyloxycarbonyl,aryloxycarbonyl, (cycloloweralkyl)oxycarbonyl, aralkyloxycarbonyl,heteroaryloxycarbonyl, heteroaralkyloxycarbonyl,(heterocycloloweralkyl)oxycarbonyl, loweralkylsulfinyl,loweralkylsulfonyl, loweralkylthio, arylthio, loweralkylcarbonyloxy,arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy,heteroaralkylcarbonyloxy, (cycloloweralkyl)carbonyloxy,(heterocycloloweralkyl)carbonyloxy, aminocarbonyl,loweraklylaminocarbonyl, arylaminocarbonyl, aralkylaminocarbonyl,heteroarylaminocarbonyl, and heteroaralkylaminocarbonyl. Examples ofparticularly useful substituents are provided above.

In a second aspect, the present invention provide compounds having thegeneral structure shown below (Compound 2):

and their pharmaceutically acceptable salts. X₃ and X₄ are selectedindependently from the group consisting of nitrogen and oxygen such thatif one of X₃ and X₄ is nitrogen, then the other of X₃ and X₄ is oxygento form thereby an isoxazole ring structure, Thus Compound 2 above willbe recognized to include the general structures:

X₅ is —(X₁₀)_(n)—, wherein n is an integer between 1 and 3 and X₁₀, foreach value of n, is selected independently from the group consisting ofoxygen, —SO_(x)— where x is and integer between 0 and 2, nitrogen,nitrogen substituted with optionally substituted loweralkyl, aryl,aralkyl, heteroaryl, heteroaralkyl, arylcarbonyl, alkylcarbonyl,aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, andmethylene or methine, each optionally substituted from the groupconsisting of halo, cyano, nitro, thio, amino, carboxyl, formyl, andoptionally substituted loweralkyl, loweralkylcarbonyloxy,arylcarbonyloxy, heteroarylcarbonyloxy, cycloalkylcarbonyloxy,cycloheteroalkylcarbonyloxy, aralkycarbonyloxy,heteroaralkylcarbonyloxy, (cycloalkyl)alkylcarbonyloxy,(cycloheteroalkyl)alkylcarbonyloxy, loweralkylcarbonyl, arylcarbonyl,heteroarylcarbonyl, cycloalkylcarbonyl, cycloheteroalkylcarbonyl,aralkycarbonyl, heteroaralkylcarbonyl, (cycloalkyl)alkylcarbonyl,(cycloheteroalkyl)alkylcarbonyl, loweralkylaminocarbonyl,arylaminocarbonyl, aralkylaminocarbonyl, heteroarylaminocarbonyl,heteroaralkylaminocarbonyl, loweralkylcarbonylamino, arylcarbonylamino,heteroarylcarbonylamino, cycloalkylcarbonylamino,cycloheteroalkylcarbonylamino, aralkylcarbonylamino,heteroaralkylcarbonylamino, (cycloalkyl)alkylcarbonylamino,(cycloheteroalkyl)alkylcarbonylamino, loweralkylamino, arylamino,aralkylamino, heteroarylamino, heteroaralkylamino, loweralkylsulfonyl,arylsulfonyl, heteroarylsulfonyl, cycloalkylsulfonyl,cycloheteroalkylsulfonyl, aralkylsulfonyl, heteroaralkylsulfonyl,(cycloalkyl)alkylsulfonyl, (cycloheteroalkyl)alkylsulfonyl,loweralkylsulfinyl, arylsulfinyl, heteroarylsulfinyl,cycloalkylsulfinyl, cycloheteroalkylsulfinyl, aralkylsulfinyl,heteroaralkylsulfinyl, (cycloalkyl)alkylsulfinyl,(cycloheteroalkyl)alkylsulfinyl, loweralkyloxy, aryloxy, heteroaryloxy,cycloalkyloxy, cycloheteroalkyloxy, aralkyloxy, heteroaralkyloxy,(cycloalkyl)alkyloxy, and (cycloheteroalkyl)alkyloxy, loweralkylthio,arylthio, heteroarylthio, cycloalkylthio, cycloheteroalkylthio,aralkylthio, heteroaralkylthio, (cycloalkyl)alkylthio,(cycloheteroalkyl)alkylthio, loweralkylthiocarbonyl, arylthiocarbonyl,heteroarylthiocarbonyl, cycloalkylthiocarbonyl,cycloheteroalkylthiocarbonyl, aralkythiocarbonyloxlthiocarbonyl,heteroaralkylthiocarbonyl, (cycloalkyl)alkylthiocarbonyl,(cycloheteroalkyl)alkylthiocarbonyl, heteroarylcarbonylthio,cycloalkylcarbonylthio, cycloheteroalkylcarbonylthio,aralkycarbonylthiooxycarbonylthio, heteroaralkylcarbonylthio,(cycloalkyl)alkylcarbonylthio, (cycloheteroalkyl)alkylcarbonylthio,loweralkyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,cycloalkyloxycarbonyl, cycloheteroalkyloxycarbonyl,aralkyoxycarbonyloxloxycarbonyl, heteroaralkyloxycarbonyl,(cycloalkyl)alkyloxycarbonyl, (cycloheteroalkyl)alkyloxycarbonyl,iminoloweralkyl, iminocycloalkyl, iminocycloheteroalkyl, iminoaralkyl,iminoheteroaralkyl, (cycloalkyl)iminoalkyl, and(cycloheteroalkyl)iminoalkyl. X₄-X₉ are selected independently from thegroup consisting of oxygen, sulfur, sulfinyl, nitrogen, and optionallysubstituted methine, and R₄ is selected from the group consisting ofoptionally substituted loweralkyl, aryl, heteroaryl, cycloalkyl,cycloheteroalkyl, aralkyl, heteroaralkyl, (cycloalkyl)alkyl, and(cycloheteroalkyl)alkyl.

Some embodiments of the present invention include those structureshaving the general form shown in Compound 2 above for which n is 1 andX₁₀ is selected from the group consisting of nitrogen, optionallysubstituted nitrogen, and optionally substituted methylene or methine.Such embodiments will be recognized as including ring systems that arecompletely delocalized as well as ring systems that are not completelydelocalized. More specific embodiments include those structures ofCompound 2 for which n is 1 and X₁₀ is selected from the groupconsisting of nitrogen, optionally substituted nitrogen, and optionallysubstituted methylene or methine and R₄ is selected from the groupconsisting of optionally substituted aryl, heteroaryl, aralkyl, andheteroaralkyl. Still more specific embodiments are those having thesubstitution pattern just described and for which R₄ is optionallysubstituted aryl or aralkyl. Yet more specific embodiments of theinventions include compounds wherein n is 1 and X₁₀ is selected from thegroup consisting of nitrogen, optionally substituted nitrogen, andoptionally substituted methylene or methine, R₄ is optionallysubstituted aryl or aralkyl, and R₄ includes at least one hydroxyl,thio, or optionally substituted loweralkyloxy, aryloxy, heteroaryloxy,loweralkylthio, arylthio, heteroarylthio, loweralkylcarbonyl,arylcarbonyl, or heteroarylcarbonyl moiety. In some embodiments havingthe structure of Compound 2 above, n is 1 and X₁₀ is selected from thegroup consisting of nitrogen, optionally substituted nitrogen, andoptionally substituted methylene or methine, R₄ is selected from thegroup consisting of phenyl, phenyloxyloweralkyl, and phenylloweralkylwherein R₄ is optionally substituted aryl or aralkyl, R₄ includes atleast one hydroxyl, thio, or optionally substituted loweralkyloxy,aryloxy, heteroaryloxy, loweralkylthio, arylthio, heteroarylthio,loweralkylcarbonyl, arylcarbonyl, or heteroarylcarbonyl moiety and R₄ isfurther substituted optionally with a moiety selected from the groupconsisting of halogen, loweralkyl, halolowerlalkyl, loweralkyloxy,halolowerlakyloxy, carboxy, loweralkyloxycarbonyl, aryloxycarbonyl,(cycloloweralkyl)oxycarbonyl, aralkyloxycarbonyl, heteroaryloxycarbonyl,heteroaralkyloxycarbonyl, (heterocycloloweralkyl)oxycarbonyl,loweralkylsulfinyl, loweralkylsulfonyl, loweralkylthio, arylthio,loweralkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy,heteroarylcarbonyloxy, heteroaralkylcarbonyloxy,(cycloloweralkyl)carbonyloxy, (heterocycloloweralkyl)carbonyloxy,aminocarbonyl, loweralkylaminocarbonyl, arylaminocarbonyl,aralkylaminocarbonyl, heteroarylaminocarbonyl, cyano, nitro, amino,loweralkylamino, and heteroaralkylaminocarbonyl.

In another embodiment, the present invention provides compounds havingthe structure of Compound 2 above for which n is 2 and each X₁₀ isselected independently from the group consisting of nitrogen, optionallysubstituted nitrogen, optionally substituted methylene, and optionallysubstituted methine. More specific embodiments include those for which nis 2 and each X₁₀ is selected independently from the group consisting ofnitrogen, optionally substituted nitrogen, optionally substitutedmethylene, and optionally substituted methine and R₄ is selected fromthe group consisting of optionally substituted aryl, heteroaryl,aralkyl, and heteroaralkyl. Also provided are embodiments in which n is2 and each X₁₀ is selected independently from the group consisting ofnitrogen, optionally substituted nitrogen, optionally substitutedmethylene, and optionally substituted methine and R₄ is optionallysubstituted aryl or aralkyl. Yet more specific embodiments having thislatter substitution pattern are those for which R₄ includes at least onehydroxyl, thio, or optionally substituted loweralkyloxy, aryloxy,heteroaryloxy, loweralkylthio, arylthio, heteroarylthio,loweralkylcarbonyl, arylcarbonyl, or heteroarylcarbonyl moiety. Stillmore particular embodiments are those wherein n is 2 and each X₁₀ isselected independently from the group consisting of nitrogen, optionallysubstituted nitrogen, optionally substituted methylene, and optionallysubstituted methine and R₄ is optionally substituted aryl or aralkyl, R₄is selected from the group consisting of phenyl, phenyloxyloweralkyl,and phenylloweralkyl and R₄ includes at least one hydroxyl, thio, oroptionally substituted loweralkyloxy, aryloxy, heteroaryloxy,loweralkylthio, arylthio, heteroarylthio, loweralkylcarbonyl,arylcarbonyl, or heteroarylcarbonyl moiety. Other, more particularembodiments are those in which n is 2 and each X₁₀ is selectedindependently from the group consisting of nitrogen, optionallysubstituted nitrogen, optionally substituted methylene, and optionallysubstituted methine, R₄ is selected from the group consisting of phenyl,phenyloxyloweralkyl, and phenylloweralkyl, R₄ includes at least onehydroxyl, thio, or optionally substituted loweralkyloxy, aryloxy,heteroaryloxy, loweralkylthio, arylthio, heteroarylthio,loweralkylcarbonyl, arylcarbonyl, or heteroarylcarbonyl moiety and R₄ isfurther substituted optionally with a moiety selected from the groupconsisting of halogen, loweralkyl, halolowerlalkyl, loweralkyloxy,halolowerlakyloxy, carboxy, loweralkyloxycarbonyl, aryloxycarbonyl,(cycloloweralkyl)oxycarbonyl, aralkyloxycarbonyl, heteroaryloxycarbonyl,heteroaralkyloxycarbonyl, (heterocycloloweralkyl)oxycarbonyl,loweralkylsulfinyl, loweralkylsulfonyl, loweralkylthio, arylthio,loweralkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy,heteroarylcarbonyloxy, heteroaralkylcarbonyloxy,(cycloloweralkyl)carbonyloxy, (heterocycloloweralkyl)carbonyloxy,aminocarbonyl, loweraklylaminocarbonyl, arylaminocarbonyl,aralkylaminocarbonyl, heteroarylaminocarbonyl, cyano, nitro, amino,loweralkylamino, and heteroaralkylaminocarbonyl.

Still other embodiments of the present invention include compounds ofthe general formula of Compound 2 for which X₄ -X₉ are selectedindependently from the group consisting of nitrogen and optionallysubstituted methine. More particular embodiments are those for whichX₆-X₉ are selected independently from the group consisting of nitrogenand optionally substituted methine and at least one of X₆ -X₉ is methinesubstituted with a moiety selected from the group consisting ofloweralkyloxy, aryloxy, heteroaryloxy, loweralkylthio, arylthio,heteroarylthio, loweralkylcarbonyl, arylcarbonyl, andheteroarylcarbonyl. Still more particular embodiments having thestructural pattern just described include those in which X₇ is methinesubstituted with hydroxy or loweralkyloxy.

In other embodiments of the invention having the general formula ofCompound 2, X₆ -X₉ are selected independently from the group consistingof nitrogen and optionally substituted methine, n is 1 and X₁₀ isselected from the group consisting of nitrogen, optionally substitutednitrogen, and optionally substituted methylene or methine. More specificembodiments are those in which X₆-X₉ are selected independently from thegroup consisting of nitrogen and optionally substituted methine, n is 1and X₁₀ is selected from the group consisting of nitrogen, optionallysubstituted nitrogen, and optionally substituted methylene or methine,and R₄ is selected from the group consisting of optionally substitutedaryl, heteroaryl, aralkyl, and heteroaralkyl. Still more specificembodiments include those for which X₆-X₉, n, and X₁₀ have the valuesjust defined and R₄ is optionally substituted aryl or aralkyl. In yetmore specific embodiments, X₆-X₉, n and X₁₀ have the values justdefined, R₄ is optionally substituted aryl or aralkyl, and R₄ includesat least one hydroxyl, thio, or optionally substituted loweralkyloxy,aryloxy, heteroaryloxy, loweralkylthio, arylthio, heteroarylthio,loweralkylcrbonyl, arylcarbonyl, or heteroarylcarbonyl moiety. Otherembodiments are those for which X₆-X₉, n, X₁₀ and R₄ have the values andsubstituents just defined, but more specifically R₄ is selected from thegroup consisting of phenyl, phenyloxyloweralkyl, and phenylloweralkyl.Still more embodiments are those for which X₆-X₉, n, X₁₀ and R₄ have thevalues and substituents just defined and R₄ is further substitutedoptionally with a moiety selected from the group consisting of halogen,loweralkyl, halolowerlalkyl, loweralkyloxy, halolowerlakyloxy, carboxy,loweralkyloxycarbonyl, aryloxycarbonyl, (cycloloweralkyl)oxycarbonyl,aralkyloxycarbonyl, heteroaryloxycarbonyl, heteroaralkyloxycarbonyl,(heterocycloloweralkyl)oxycarbonyl, loweralkylsulfinyl,loweralkylsulfonyl, loweralkylthio, arylthio, loweralkylcarbonyloxy,arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy,heteroaralkylcarbonyloxy, (cycloloweralkyl)carbonyloxy,(heterocycloweralkyl)carbonyloxy, aminocarbonyl,loweraklylaminocarbonyl, arylaminocarbonyl, aralkylaminocarbonyl,heteroarylaminocarbonyl, cyano, nitro, amino, loweralkylamino, andheteroaralkylaminocarbonyl.

Yet other embodiments of the invention including the compounds of thegeneral formula of Compound 2 are those in which X₆-X₉ are selectedindependently from the group consisting of nitrogen and optionallysubstituted methine, n is 2 and each X₁₀ is selected independently fromthe group consisting of nitrogen, optionally substituted nitrogen,optionally substituted methylene, and optionally substituted methine.More specific embodiments are those in which X₆-X₉ are selectedindependently from the group consisting of nitrogen and optionallysubstituted methine, n is 2 and each X₁₀ is selected independently fromthe group consisting of nitrogen, optionally substituted nitrogen,optionally substituted methylene, and optionally substituted methine,and R₄ is selected from the group consisting of optionally substitutedaryl, heteroaryl, aralkyl, and heteroaralkyl. Still more specificembodiments include those for which X₆-X₉, n, and X₁₀ have the valuesjust defined and R₄ is optionally substituted aryl or aralkyl. In yetmore specific embodiments, X₆-X₉, n, X₁₀ and R₄ have the values justdefined and R₄ includes at least one hydroxyl, thio, or optionallysubstituted loweralkyloxy, aryloxy, heteroaryloxy, loweralkylthio,arylthio, heteroarylthio, loweralkylcarbonyl, arylcarbonyl, orheteroarylcarbonyl moiety. Yet more specific embodiments are those forwhich X₆-X₉, n, X₁₀ and R₄ have the values and substituents justdefined, but more particularly in which R₄ is selected from the groupconsisting of phenyl, phenyloxyloweralkyl, and phenylloweralkyl. Otherembodiments having the general structure of Compound 2 are those forwhich X₆-X₉ are selected independently from the group consisting ofnitrogen and optionally substituted methine, n is 2 and each X₁₀ isselected independently from the group consisting of nitrogen, optionallysubstituted nitrogen, optionally substituted methylene, and optionallysubstituted methine, R₄ is selected from the group consisting of phenyl,phenyloxyloweralkyl, and phenylloweralkyl, R₄ includes at least onehydroxyl, thio, or optionally substituted loweralkyloxy, aryloxy,heteroaryloxy, loweralkylthio, arylthio, heteroarylthio,loweralkylcarbonyl, arylcarbonyl, or heterorylcarbonyl moiety, and R₄ isfurther substituted optionally with a moiety selected from the groupconsisting of halogen, loweralkyl, halolowerlalkyl, loweralkyloxy,halolowerlakyloxy, carboxy, loweralkyloxycarbonyl, aryloxycarbonyl,(cycloloweralkyl)oxycarbonyl, aralkyloxycarbonyl, heteroaryloxycarbonyl,heteroaralkyloxycarbonyl, (heterocycloloweralkyl)oxycarbonyl,loweralkylsulfinyl, loweralkylsulfonyl, loweralkylthio, arylthio,loweralkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy,heteroarylcarbonyloxy, heteroaralkylcarbonyloxy,(cycloloweralkyl)carbonyloxy, (heterocycloloweralkyl)carbonyloxy,aminocarbonyl, loweraklylaminocarbonyl, arylaminocarbonyl,aralkylaminocarbonyl, heteroarylaminocarbonyl, cyano, nitro, amino,loweralkylamino, and heteroaralkylaminocarbonyl.

4.3 Synthesis of the Compounds of the Invention

The compounds of the present invention can be synthesized usingtechniques and materials known to those of skill in the art (Carey andSundberg 1943; Carey and Sundberg 1983; Greene and Wuts 1991; March1992). Starting materials for the compounds of the invention may beobtained using standard techniques and commercially available precursormaterials, such as those available from Aldrich Chemical Co. (Milwaukee,Wis.), Sigma Chemical Co. (St. Louis, Mo.), Lancaster Synthesis(Windham, N.H.), Apin Chemicals, Ltd. (New Brunswick, N.J.), RyanScientific (Columbia, S.C.), Maybridge (Cornwall, England), Arcos(Pittsburgh, Pa.), and Trans World Chemicals (Rockville, Md.)

The procedures described herein for synthesizing the compounds of theinvention may include one or more steps of protection and deprotection(e.g., the formation and removal of acetal groups) (Green and Wuts1991). In addition, the synthetic procedures disclosed below can includevarious purifications, such as column chromatography, flashchromatography, thin-layer chromatography (“TLC”), recrystallization,distillation, high-pressure liquid chromatography (“HPLC”) and the like.Also, various techniques well known in the chemical arts for theidentification and quantification of chemical reaction products, such asproton and carbon-13 nuclear magnetic resonance (¹H and ¹³C NMR),infrared and ultraviolet spectroscopy (“IR” and “UV”), X-raycrystallography, elemental analysis (“EA”). HPLC and mass spectroscopy(“MS”) can be used for identification, quantitation and purification aswell.

Scheme 1 is a general scheme for synthesis of isoxazoles.

Step A is a Claisen-type condensation, in which X is a leaving groupsuch as —OR (R═alkyl, aryl, arlkyl, heteroaryl, or heteroaralkyl), orhalogen. When X is —OR and R is alkyl (e.g., X is methoxy or ethoxy) thereaction of 1a and 1b to produce 1c can be done using procedures knownto those of skill in the organic chemistry arts (Tietze and Eicher1989). When X is halogen, e.g., Cl, a typical procedure involvesdeprotonation of ketone 1a with a base such as lithiumbis(trimethylsilyl)amide (LiHMDS) followed by addition of 1b. Suitablesolvents for performing such reactions will be familiar to those ofskill in the organic chemistry arts. Examples of suitable solventsinclude ether-type solvents such as tetrahydrofuran (“THF”), diethylether (H₃CH₂COCH₂CH₃), or aliphatic and aromatic hydrocarbon solventssuch as cyclohexane (C₆H₁₂) and toluene (C₇H₈). Typical reactiontemperatures range from −78° C. to +25° C. and the reaction times from 6hours (“h”) to 20 h. Step B is a cycloaddition reaction to form thedesired isoxazole. In a typical procedure, a mixture of 1c, twoequivalents of hydroxyamine hydrochloride, and three equivalents ofpyridine in ethanol are heated to reflux overnight. Removal of thesolvent followed by extraction yields a crude material that can bepurified to afford substantially pure compound 1d. If R₁ and R₂ are notidentical, then a mixture of regioisomers is formed. In some cases,protecting groups have to be removed to obtain targeted compound 1e, asillustrated by Step C. Protection and deprotection will depend greatlyon the chemical properties of the molecule and its functional groups;appropriate methods for protection and deprotection are well known inthe organic chemistry arts (Greene and Wuts 1991). For example, when R₁is methoxyphenyl, three methods can be used for demethylation: 1)reaction of aqueous hydrogen bromide (HBr) and glacial acetic acid with1d with heating to 100-120° C. for 6 to 16 h; 2) reaction of ethanethiol, aluminum trichloride, and 1d in dichloroethane with stirring atroom temperature (“rt”) for 16 to 72 h; or 3) stirring boron tribromidewith 1d in dichloromethane at room temperature overnight.

Scheme 2 describes an alternative method to synthesize compound 1c ofScheme 1.

Step A above can be performed using various methods familiar to those ofskill in the organic chemistry arts. For example, at least three wellknown methods can be used to convert 2a to 1c: 1) deprotonation of 2awith a base such as sodium hydride (NaH) in an aprotic solvent such asdimethylformamide (“DMF”) or THF, followed by reaction of the resultinganion with an electrophile R₃X, wherein X is a leaving group such ashalogen or MsO; or 2) compound 2a is reacted with R₃X, potassiumcarbonate and tetrabutylammonium bromide in DMF while stirring atrt—100° C. for 6 to 24 h. If R₃ is paraalkyloxyphenyl, then a plumbatemethod can be applied (Craig, Holder et al. 1979; Pinhey, Holder et al.1979).

Regiospecific synthesis of the isoxazoles of the invention can beperformed using known methods. One example of such a method is shown inScheme 3 below (Perkins, Beam et al.):

Scheme 4 describes an alternative method to synthesize compound 1d inScheme 1.

Formation of isoxazoles 4a from 1,3-diketone 2a (Step A) can beperformed using Step B of Scheme 1. Bromination of isoxazole 4a (Step B)can be accomplished by addition of bromine in chloroform solution to asolution of 4a under temperatures from rt—55° C. for a period between0.5 to 2 h to form 4-bromoisoxazole 4b. A variety of substituents at theR₃ can be introduced to 4-bromoisoxazole 4b to form the desired product1d (Step C) as will be apparent to those of skill in the organicchemistry arts. For example, metal-halogen exchange followed by trappingthe resulting isoxazole anion with an electrophile can be used to attachR₃. This can be done, for example, by reaction of bromoisoxazole 4b inTHF solution at −78° C. with n-BuLi. The mixture is stirred at −78° C.for 1 h. The desired electrophile corresponding to R₃ is then added, andthe reaction is warmed to 0° C.—rt over a period between 2 to 16 h.Suitable electrophiles include, but art not limited to, the following:alkyl halides, disulfides, iodine, N-chlorosuccinimide, tosyl nitrile,ethyl chloroformate, acid chlorides, carbon dioxide, dimethylformamide,aldehydes, Weinreb amides and sulfonyl chlorides.

Alternatively, a 4-carboxyisoxazole (i.e., R₃═—CO₂) can be obtained ifcarbon dioxide is used as the electrophile. The carboxylic acid can befurther transformed to various esters, amides, and ketones. To form anamide at R₃, typical amide bond formation condition can be applied. Forexample, the corresponding carboxylic acid can be activated with1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (“EDC”) HCl salt,1-hydroxybenzotriazole (“HOBt”), and Hünig's base and mixed with aprimary or secondary amine in THF or DMF. The reaction is complete in 6to 16 hours at rt. Suzuki coupling can also be used to introduce aryland alkenyl moieties at R₃ (Miyaura, Author et al. 1979; Miyaura andSuzuki 1979). The Ullmann reaction can be used to introduce aryloxygroups at R₃ (Knight; Semmelback, Author et al.). Moieties having C—Nand C—O bonds at 4-position of isoxazole 4b can be achieved by applyingpalladium catalyzed coupling reactions (Palucki, Wolfe et al. 1996;Wolfe and Buchwald 1996; Wolfe, Wagaw et al. 1996).

Scheme 5 illustrates more specific modifications at 4-position of theisoxamole.

Starting material 5a can be synthesized by methods described above. Thelinker Z can be —CH₂—, —O—, —S—, —SO₂—, —NR′R″—, —(C═O)—, —(C═NOR)—, orthe aryl group can be attached to the isoxazole core directly. In theScheme above, R₃ is a phenol protecting group that can be removedselectively (Greene and Wuts 1991) as shown in Step A. The free hydroxylgroup can be derivatized using known methods and materials (Step B).However, other suitable groups such as, but not limited to, thiols,protected thiols, amines, and the like can be synthesized usinganalogous methodologies. One specific methodology is described withrespect to Scheme 6 below where Z is —SO₂— or —(C═O)— and Y is O, S, orN. The index can be 1, 2, or 3, and R₄ is —NR′R″ or —N(R′)(C═O)R″. Inone example, sodium hydride was mixed with HY(CH₂)_(n)R₄, to generatethe nucleophile and added to 6a in THF or DMF solution at a temperaturebetween rt and 60° C. and completed within 2 to 8 h.

Specific modifications at 5-position of the isoxazole can be performedusing the methodologies described with Scheme 7 below:

where E is alkyl, aryl, aralkyl, halo, cyano, amido, carboxy, sulfide,and sulfoxide. Starting material 7a can be synthesized according tomethods described above. The functional group E is introduced using themethods described in Step C of Scheme 4 above. Modifications at the4-position of the isoxazole can be made, for example, using the methodsdescribed with respect to Scheme 8.

Starting material 8a was synthesized according to methods described inScheme 1. Bromination at the methyl position was performed usingN-bromosuccinimide in carbon tetrachloride. Alkylation to formderivatives of 8c where R₄ is —OR, —SR or —NRR′ can be conducted withappropriate nucleophile in a suitable solvent (e.g., DMF or THF) attemperatures ranging between rt and 100° C.

The procedures described above can be applied to solid phasemethodologies as well. The actual implementation depends, of course, onthe details of the desired products and starting materials. One exampleof a suitable methodology, where R₁ is hydroxyphenyl, is shown in Scheme9.

In step A, commercially available hydroxylated Rink resin (Calbiochem,La Jolla, CA) is reacted with mesyl chloride and Hünig's base inmethylene chloride (CH₂Cl₂) at 0° C. with warming to room temperatureover a two-hour period. Next, 4-hydroxyacetophenone and Hünig's base arereacted with the resin product in methylene chloride at room temperatureovernight provides resin-bound ketone 9a. Reaction of the bound ketonewith an ester bearing the R₃ substituent (R₃CO₂R) and base (e.g.,potassium tert-butoxide, t-BuOK and dibenzo- 18-crown-6) in a suitablesolvent (e.g., THF) at 70° C. for six hours (Step B) provides diketone9b. Deprotonation of 9b, using, e.g., tert-butyl ammonium iodide(“TBAI”) under mild conditions (70° C. overnight) and the R₂ substituentbearing a suitable leaving group (e.g., halogen, tosylate, mesylate)provides 9c. Cyclization of 9c to form the desired isoxazole(resin-bound regioisomers 9d and 9e) can be performed by reaction of thebound diketone with H₂NOH·HCl and Hünig's base in a suitable solvent(e.g., dimethylsulfoxide, (“DMSO”)) at 70° C. for fifteen hours.Cleavage from the resin can be performed under mild conditions (e.g.,reaction with 5% trifluoroacetic acid. (“TFA”) in methylene chloride)provides the final products 9d and 9e.

4.4 Biological Activity

The activities of the compounds of the invention to function as estrogenreceptor agonists or antagonists can be determined using a wide varietyof assays known to those having skill in the biochemistry, medicinalchemistry, and endochrinology arts. Several useful assays are describedgenerally in this Section 4.4. Specific examples are described inSection 5.2 below.

4.4.1 Assays for Estrogen Receptor Modulating Activity In Vivo and ExVivo

4.4.1.1 Allen-Doisy Test for Estrogenicity

This test (described in greater detail in Section 5.2.1.1 below) is usedto evaluate a test compound for estrogenic activity, and, morespecifically, the ability of a test compound to induce an estrogeniccornification of vaginal epithelium (Allen and Doisy 1923; Mühlbock1940; Terenius 1971). Test compounds are formulated and administeredsubcutaneously to mature, ovariectomized female rats in test groups. Inthe third week after bilateral ovariectomy, the rats are primed with asingle subcutaneous dose of estradiol to ensure maintenance ofsensitivity and greater uniformity of response. In the fourth week, 7days after priming, the test compounds are administered. The compoundsare given in three equal doses over two days (evening of the first dayand morning and evening of the second day). Vaginal smears are thenprepared twice daily for the following three days. The extent ofcornified and nucleated epithelial cells, as well as of leucocytes areevaluated for each of the smears.

4.4.1.2 Anti-Allen-Doisy Test for Anti-Estrogenicity

This test (described in greater detail in Section 5.2.1.2 below) is usedto evaluate a test compound for anti-estrogenic activity by observationof cornification of the vaginal epithelium of in ovariectomized ratsafter administration of a test compound (Allen and Doisy 1923; Mühlbock1940; Terenius 1971). Evaluation of anti-estrogenic activity isperformed using mature female rats which, two weeks after bilateralovariectomy, are treated with estradiol to induce a cornification of thevaginal epithelial. This was followed by administration of the testcompound in a suitable formulation daily for 10 days. Vaginal smears areprepared daily, starting on the first day of test compoundadministration and proceeding until one day following the lastadministration of test compound. The extent of cornified and nucleatedepithelial cells and leucocytes is evaluated for each of the smears asabove.

4.4.1.3 Immature Rat Uterotrophic Bioassay for Estrogenicity andAnti-Estrogenicity

Changes in uterine weight in response to estrogenic stimulation can beused to evaluate the estrogenic characteristics of test compounds onuterine tissues (Reel, Lamb et al., 1996; Ashby, Odum et al. 1997). Inone example, described in Section 5.2.1.3 below, immature female ratshaving low endogenous levels of estrogen are dosed with test compound(subcutaneously) daily for 3 days. Compounds are formulated asappropriate for subcutaneous injection. As a control, 17-beta-estradiolis administered alone to one dose group. Vehicle control dose groups arealso included in the study. Twenty-four hours after the last treatment,the animals are necropsied, and their uteri excised, nicked, blotted andweighted to. Any statistically significant increases in uterine weightin a particular dose group as compared to the vehicle control groupdemonstrate evidence of estrogenicity.

4.4.1.4 Estrogen Receptor Antagonist Efficacy In MCF-7 Xenograft Model

This test (described in detail in Section 5.2.1.4 below) is used toevaluate the ability of a compound to antagonize the growth of anestrogen-dependent breast MCF-7 tumor in vivo. Female Ncr-nu mice areimplanted subcutaneously with an MCF-7 mammary tumor for an existing invivo passage. A 17-β-estradiol pellet is implanted on the side oppositethe tumor implant on the same day. Treatment with test compound beginswhen tumors have reached a certain minimum size (e.g., 75-200 mg). Thetest compound is administered subcutaneously on a daily basis and theanimals are subjected to daily mortality checks. Body weights and tumorvolume are determined twice a week starting the first day of treatment.Dosing continues until the tumors reach 1,000 mm³. Mice with tumorslarger than 4,000 mg, or with ulcerated tumors, are sacrificed prior tothe day of the study determination. The tumor weights of animals in thetreatment group are compared to those in the untreated control group aswell as those given the estradiol pellet alone.

4.4.1.5 OVX Rat Model

This model evaluates the ability of a compound to reverse the decreasein bone density and increase in cholesterol levels resulting fromovariectomy. One example of such a model is described in Section5.2.1.5. Three-month old female rats are ovariectomized, and testcompounds are administered daily by subcutaneous route beginning one daypost-surgery. Sham operated animals and ovariectomized animals withvehicle control administered are used as control groups. After 28 daysof treatment, the rats are weighed, the overall body weight gainsobtained, and the animals euthanized. Characteristics indicative ofestrogenic activity, such as blood bone markers (e.g., osteocalcin,bone-specific alkaline phosphatase), total cholesterol, and urinemaskers (e.g., deoxypyridinoline, creatinine) are measured in additionto uterine weight. Both tibiae and femurs are removed from the testanimals for analysis, such as the measurement of bone mineral density. Acomparison of the ovariectomized and test vehicle animals to the shareand ovariectomized control animals allows a determination of the tissuespecific estrogenic/anti-estrogenic effects of the test compounds.

4.4.2 Assays for Estrogen Receptor Modulating Activity in Vitro

4.4.2.1 ERα/ERβ Binding Assays

For evaluation of ERα/ERβ receptor binding affinity, a homogeneousscintillation proximity assays is used (described in Sections 5.2.2.1and 5.2.2.2 below). 96-well plates are coated with a solution of eitherERα or ERβ. After coating, the plates are washed with PBS. The receptorsolution is added to the coated plates, and the plates are incubated.For library screening, [³H]estradiol is combined with the test compoundsin the wells of the 96-well plate. Non-specific binding of theradio-ligand is determined by adding estradiol to one of the wells as acompetitor. The plates are gently shaken to mix the reagents and asample from each of the wells is then transferred to the pre-coated ERαor ERβ plates. The plates are sealed and incubated, and thereceptor-bound estradiol read directly after incubation using ascintillation counter to determine test compound activity. If estimatesof both bound and free ligand are desired, supernatant can be removedand counted separately in a liquid scintillation counter.

4.4.2.2 ERα/ERβ Transaction Assays

The estrogenicity of the compounds of the invention can be evaluated inan in vitro bioassay using Chinese hamster ovary (“CHO”) cells that havebeen stably co-transfected with the human estrogen receptor (“hER”), therat oxytocin promoter (“RO”) and the luciferase reporter gene (“LUC”) asdescribed in Section 5.2.2.3 below. The estrogen transactivationactivity (potency ratio) of a test compound to inhibit transactivationof the enzyme luciferase as mediated by the estrogen receptor iscompared with a standard and the pure estrogen autogonist.

4.4.2.3 MCF-7 Cell Proliferation Assays

MCF-7 cells are a common line of breast cancer cells used to determinein vitro estrogen receptor agonist/antagonist activity (MacGregor andJordan 1998). The effect of a test compound on the proliferation ofMCF-7 cells, as measured by the incorporation of 5-bromo-2′-deoxyuridine(“BrdU”) in a cheniluminescent assay format, can be used to determinethe relative agonist/antagonist activity of the test compound, MCF-7cells (ATCC HTB-22) are maintained in log-phase culture. The cells areplated and incubated in phenol-five medium to avoid external sources ofestrogenic stimulus (MacGregor and Jordan 1998). The test compound isadded at varying concentration to determine an IC₅₀, for the compound.To determine agonist activity, the assay system is kept free of estrogenor estrogen-acting sources. To determine antagonist activity, controlledamounts of estrogen are added.

4.5 Pharmaceutical Compositions

The compounds of the present invention can be used in the form of saltsderived from inorganic or organic acids. These salts include, but arenot limited to, the following: acetate, adipate, alginate, citrate,aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate,camphorsulfonate, digluconate, cyclopentanepro-pionate, dodecylsulfate,ethanesulfonate, glucoheptanoate, glycerophosphate, hemi-sulfate,heptanoate, hexanoate, fumarate, hydrochloride, hydrodromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, nicotinate, 2-naphth-alenesulfonate, oxalate, pamoate,pectinate, sulfate, 3-phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, p-toluenesulfonate and undecanoate. Also, any basicnitrogen-containing groups can be quaternized with agents such asloweralkyl halides, such as methyl, ethyl, propyl, and butylchloride,bromides, and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl,and diamyl sulfates, long chain halides such as decyl, lauryl, myristyland stearyl chlorides, bromides and iodides, aralkyl halides like benzyland phenethyl bromides, and others. Water or oil-soluble or dispersibleproducts are thereby obtained.

Examples of acids which may be employed to form pharmaceuticallyacceptable acid addition salts include such inorganic acids ashydrochloric acid, sulfuric acid, and phosphoric acid, and organic acidssuch as oxalic acid, maleic acid, succinic acid and citric acid. Basicaddition salts can be prepared in situ during the final isolation andpurification of the compounds of the invention, or separately byreacting carboxylic acid moieties with a suitable base such as thehydroxide, carbonate or bicarbonate of a pharmaceutically acceptablemetal cation or with ammonia, or an organic primary, secondary ortertiary amine. Pharmaceutically acceptable salts include, but are notlimited to, cations based on the alkali and alkaline earth metals, suchas sodium, lithium, potassium, calcium, magnesium, aluminum salts andthe like, as well as nontoxic ammonium, quaternary ammonium, and aminecations, including but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamino, ethylamine, and the like. Other representative organicamines useful for the formation of base addition salts includediethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazineand the like.

Compounds of the present invention can be administered in a variety ofways including enteral, parenteral and topical routes of administration.For example, suitable modes of administration include oral,subcutaneous, transdermal, transmucosal, iontophoretic, intravenous,intramuscular, intraperitoneal, intranasal, subdural, rectal, vaginal,and the like.

In accordance with other embodiments of the present invention, there isprovided a composition comprising an estrogen receptor-modulatingcompound of the present invention, together with a pharmaceuticallyacceptable carrier or excipient.

Suitable pharmaceutically acceptable excipients include processingagents and drug delivery modifiers and enhancers, such as, for example,calcium phosphate, magnesium stearate, talc, monosaccharides,disaccharides, starch, gelatin, cellulose, methyl cellulose, sodiumcarboxymethyl cellulose, dextrose, hydroxypropyl-β-cyclodextrin,polyvinylpyrrolidinone, low melting waxes, ion exchange resins, and thelike, as well as combinations of any two or move thereof. Other suitablepharmaceutically acceptable excipients are described in Remington'sPharmaceutical Sciences, Mack Pub. Co., N.J. (1991), which isincorporated herein by reference.

Pharmaceutical compositions containing estrogen receptor modulatingcompounds of the present invention may be in any form suitable for theintended method of administration, including, for example, a solution, asuspension, or an emulsion. Liquid carriers are typically used inpreparing solutions, suspensions, and emulsions. Liquid carrierscontemplated for use in the practice of the present invention include,for example, water, saline, pharmaceutically acceptable organicsolvent(s), pharmaceutically acceptably oils or fats, and the like, aswell as mixtures of two or more thereof. The liquid carrier may containother suitably pharmaceutically acceptable additives such assolubilizers, emulsifiers, nutrients, buffers, preservatives, suspendingagents, thickening agents, viscosity regulators, stabilizers, and thelike. Suitable organic solvents include, for example, monohydricalcohols, such as ethanol, and polyhydric alcohols, such as glycols.Suitable oils include, for example, soybean oil, coconut oil, olive oil,safflower oil, cottonseed oil, and the like. For parenteraladministration, the carrier can also be an oily ester such as ethyloleate, isopropyl myristate, and the like. Compositions of the presentinvention may also be in the form of microparticles, microcapsules,liposomal encapsulates, and the like, as well as combinations of any twoor more thereof.

The compounds of the present invention may be administered orally,parenterally, sublingually, by inhalation spray, rectally, vaginally, ortopically in dosage unit formulations containing conventional nontoxicpharmaceutically acceptable carriers, adjuvants, and vehicles asdesired. Topical administration may also involve the use of transdermaladministration such as transdermal patches or ionophoresis devices. Theterm parenteral as used herein includes subcutaneous injection,intravenous, intramuscular, intrasternal injection, or infusiontechniques.

Injectable preparation, for example, sterile injectable aqueous oroleginous suspensions may be formulated according to the known art usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a nontoxic parenterally acceptable diluent or solvent, forexample, as a solution in 1,3-propanediol. Among the acceptable vehiclesand solvents that may be employed are water, Ringer's solution, andisotonic sodium chloride solution. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid can beuseful in the preparation of injectables.

Suppositories for rectal or vaginal administration of the drug can beprepared by mixing the drug with a suitable nonirritating excipient suchas cocoa butter and polyethylene glycols that are solid at ordinarytemperatures but liquid at the rectal temperature and will thereforemelt in the rectum and release the drug.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage form, theactive compound may be admixed with at least one inert diluent such assucrose lactose or starch. Such dosage forms may also comprise, as isnormal practice, additional substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets, and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspension, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, cyclodextrins, and sweetening,flavoring, and perfuming agents.

The compounds of the present invention can also be administered in theform of liposomes. As is known in the art, liposomes are generallyderived from phospholipids or other lipid substances. Liposomes areformed by mono- or multilamellar hydrated liquid crystals that aredispersed in an aqueous medium. Any non-toxic, physiologicallyacceptable and metabolizable lipid capable of forming liposomes can beused. The present compositions in liposome form can contain, in additionto a compound of the present invention, stabilizers, preservatives,excipients, and the like. The preferred lipids are the phospholipids andphosphatidyl cholines (lecithins), both natural and synthetic. Methodsto form liposomes are known in the art (Prescott 1976).

While the compounds of the invention can be administered as the activepharmaceutical agent, they can also be used in combination with one ormore other compound as described herein, and/or in combination withother agents used in the treatment and/or prevention of estrogenreceptor-mediated disorders. Alternatively, the compounds of the presentinvention can be administered sequentially with one or more such agentsto provide sustained therapeutic and prophylactic effects. Suitableagents include, but are not limited to, other SERMs as well astraditional estrogen agonists and antagonists. Representative agentsuseful in combination with the compounds of the invention for thetreatment of estrogen receptor-mediated disorders include, for example,tamoxifen, 4-hydroxytamoxifen, raloxifene, toremifene, droloxifene,TAT-59, idoxifene, RU 58,688, EM 139, ICI 164,384, ICI 182,780,clomiphene, MER-25, DES, nafoxidene, CP-336,156, GW5638, LY139481,LY353581, zuclomiphene, enclomiphene, ethamoxytriphetol, delmadinoneacetate, bisphosphonate, and the like. Other agents that can be combinedwith one or more of the compounds of the invention include aromataseinhibition such as, but not limited to, 4-hydroxyandrostendione,plomestane, exemestane, aminogluethimide, rogletimide, fadrozole,fadrozole, vorozole, letrozole, and anastrozole.

Still other agents useful for combination with the compounds of theinvention include, but are not limited to antineoplastic agents, such asalkylating agents. Other classes of relevant antineoplastic agentsinclude antibiotics, hormonal antineoplastics and antimetabolites.Examples of useful alkylating agents include alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines, such as a benzodizepa,carboquone, meturedepa and uredepa; ethylenimines and methylmelaminessuch as altretamide, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylolmelamine; nitrogen mustardssuch as chlorambucil, chlornaphazine, cyclophosphamide, estramustine,iphosphamide, mechlorethanine, mechlorethamine oxide hydrochloride,melphtalan, novembichine, phenesterine, prednimustine, trofosfamide, anduracil mustard; nitroso urea, such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine, dacarbazine,mannomustine, mitobronitol, mitolactol, mitolactol and pipobroman. Moresuch agents will be known to those having skill in the medicinalchemistry and oncology arts.

Additional agents suitable for combination with the compounds of thepresent invention include protein synthesis inhibitors such as abrin,aurintricarboxylic acid, chloramphenicol, colicin E3, cycloheximide,diphtheria toxin, edeine A, emetine, erythromycin, ethionine, fluoride,5-fluorotryptophan, fusidic acid, guanylyl methylene diphosphonate andguanylyl imidodiphosphate, kanamycin, kasugamycin, kasugamycin,kirromycin, and O-methyl threonine, modeccin, neomycin, norvaline,pactanycin, paromomycine, puromycin, ricin, α-sarcin, shiga toxin,showdomycin, sparsomycin, spectinomycin, streptomycin, tetracycline,thiostrepton and trimethoprin, Inhibitors of DNA synthesis, includingalkylating agents such as dimethyl sulfate, mitomycin C, nitrogen andsulfur mustards, MNNG and NMS; intercalating agents such as acridinedyes, actinomycins, adrianycin, anthracenes, benzopyrene, ethidiumbromide, propidium diiodide-intertwining, and agents such as distamycinand netropsin, can also be combined with compounds of the presentinvention in pharmaceutical compositions. DNA base analogs such asacyclovir, adenine, β-1-D-arabinoside, amethopterin, aminopterin,2-aminopurine, aphidiolin, 8-azaguanine, azaserine, 6-azauracil,2′-azido-2′-deoxynucleosides, 5-bromodeoxycytidine, cytsine,β-1-D-arabinoside, diazooxynorleucine, dideoxynucleosides,5-fluorodeoxycytidine, 5-fluorodeoxyuridine, 5-fluorouracil, hydroxyureaand 6-mercaptopurine also can be used in combination therapies with thecompounds of the invention. Topoisomerase inhibitors, such ascoumermycin, nalidixic acid, novobiocin and oxolinic acid, inhibitors ofcell division, including colcemide, colchicine, vinblastine andvincristine, and RNA synthesis inhibitors including actinomycin D,α-amanitine and other fungal amatoxins, cordycepin (3′-deoxyadenosine),dichloroibofuranosyl benzimidazole, rifampicine, streptovarin andstreptolydigin also can be combined with the compounds of the inventionto provide pharmaceutical compositions. Still more such agents will beknown to those having skill in the medicinal chemistry and oncologyarts.

In addition, the compounds of the present invention can be used, eithersingly or in combination as described above, in combination with othermodalities for preventing or treating estrogen receptor-medicateddisease or disorders. Such other treatment modalities include withoutlimitation, surgery, radiation, hormone supplementation, and dietregulation. These can be performed sequentially (e.g., treatment with acompound of the invention following surgery or radiation) or incombination (e.g., in addition to a diet reginen).

In another embodiment, the present invention includes compounds andcompositions in which a compound of the invention is either combinedwith, or covalently bound to, a cytotoxic agent bound to a targetingagent, such as a monoclonal antibody (e.g., or humanized monoclonalantibody). It will be appreciated that the latter combination may allowthe introduction of cytotoxic agents into cancer cells with greaterspecifically. Thus, the active form of the cytotoxic agent (i.e., thefree form) will be present only in cells targeted by the antibody. Ofcourse, the compounds of the invention may also be combined withmonoclonal antibodies that have therapeutic activity against cancer.

The additional active agents may generally be employed is therapeuticamounts as indicated in the PHYSICIANS' DESK REFERENCE (PDR) 53rdEdition (1999), which is incorporated herein by reference, or suchtherapeutically useful amounts as would be known to one of ordinaryskill in the art. The compounds of the invention and the othertherapeutically active agents can be administered at the recommendedmaximum clinical dosage or a lower doses. Dosage levels of the activecompounds in the compositions of the invention may be varied in obtain adesired therapeutic response depending on the route of administration,severity of the disease and the response of the patient. The combinationcan be administered as separate compositions or as a single dosage formcontaining both agents. When administered as a combination, thetherapeutic agents can be formulated as separate compositions that aregiven at the same time or different times, or the therapeutic agents canbe given as a single composition.

4.6 Treatment of Estrogen Receptor-Mediated Disorders

In accordance with the yet other embodiments, the present inventionprovides methods for treating or preventing an estrogenreceptor-mediated disorder in a human or animal subject in which anamount of an estrogen receptor-modulating compounds of the inventionthat is effective to modulate estrogen receptor activity in the subject.Other embodiments provided methods for treating a cell or a estrogenreceptor-mediated disorder in a human or animal subject, comprisingadministering to the cell or to the human or animal subject an amount ofa compound or composition of the invention effective to modulateestrogen receptor activity in the cell or subject. Preferably, thesubject will be a human or non-human animal subject. Modulation ofestrogen receptor activity detectable suppression or up-regulation ofestrogen receptor activity either as compared to a control or ascompared to expected estrogen receptor activity.

Effective amounts of the compound of the invention generally include anyamount sufficient to detectably modulate estrogen receptor activity byany of the assays described herein, by other activity assays known tothose having ordinary skill in the art, or by detecting prevention oralleviation of symptoms in a subject afflicted with a estrogenreceptor-mediated disorder.

Estrogen receptor-mediated disorders that may be treated in accordancewith the invention include any biological or medical disorder in whichestrogen receptor activity in implicated or in which the inhibition ofestrogen receptor potentiates or retards signaling through a pathwaythat is characteristically defective in the disease to be treated. Thecondition or disorder may either be caused or characterized by abnormalestrogen receptor activity. Representative estrogen receptor-mediateddisorders include, for example, osteoporosis, atheroschlerosis,estrogen-mediated cancers (e.g., breast and endometrial cancer).Turner's syndrome, benign prostate hyperplasia (i.e., prostateenlargement), prostate cancer, elevated cholesterol, restenosis,endometriosis, uterine fribroid disease, skin and/or vagina atrophy, andAlzheimer's disease. Successful treatment of a subject in accordancewith the invention may result in the prevention, inducement of areduction in, or alleviation of symptoms in a subject afflicted with anestrogen receptor-mediated medical or biological disorder. Thus, forexample, treatment can result in a reduction in breast or endometrialtumors and/or various clinical markers associated with such cancers.Likewise, treatment of Alzheimer's disease can result in a reduction inthe rate of disease progression, detected, for example, by measuring areduction in the rate of increase of dementia.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. It will beunderstood, however, that the specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, route of administration, rate ofexcretion, drug combination, and the severity of the particular diseaseundergoing therapy. The prophylactically or therapeutically effectiveamount for a given situation can be readily determined by routineexperimentation and is within the skill and judgment of the ordinaryclinician.

For exemplary purposes of the present invention, a prophylactically ortherapeutically effective dose will generally be from about 0.1mg/kg/day to about 100 mg/kg/day, preferably from about 1 mg/kg/day toabout 20 mg/kg/day, and most preferably from about 2 mg/kg/day to about10 mg/kg/day of a estrogen receptor-modulating compound of the presentinvention, which may be administered in one or multiple doses.

EXAMPLES

The following Examples are provided to illustrate certain aspects of thepresent invention and to aid those of skill in the art in the art inpracticing the invention. These Examples are in no way to be consideredto limit the scope of the invention in any manner.

5.1 Preparation of Compounds of the invention

5.1.1 General Procedures

All reactions were carried out under nitrogen or argon atmosphere. Allreagents obtained from commercial sources were used without furtherpurification. Anhydrous solvents were obtained from commercial sourcesand used without further drying. Separation and purification of theproducts were carried out using any or combination of the followingmethods. Flash column chromatography was performed with silica gel,200-400 mesh, 60 A (Aldrich Chemical Company, Inc., Milwaukee. Wis.) ora Flash 40 chromatogrpahy system and KP-Sil, 60 A (Biotage,Charlottesville, Va.). Typical solvents employed were dichloromethane(DCM), methanol (MeOH), ethyl acetate (BtOAc), and hexane (Hex),Preparative TLC was conducted using 20×20 cm plates coated with Merch-EMType-60, GF-254 silica gel. Preparative HPLC was performed with DynamaxSystem using a C-18 reversed phase column (Ranin).

Compounds of the present invention were characterized by LC/MS usingeither Waters Micromass Platform LCZ system (ionization mode; electronspray positive; column; HP-Eclipse XDB-C18, 2×50 mm, buffer A: H₂O with0.1% trifluoroacetic acid (TFA), buffer B: acetonitrile (MeCN) with 0.1%TFA, elution gradient: 5-95% buffer over 5 minute period, flow rate: 0.8mL/min) or HP 1100 Series LC/MSD system (ionization mode: electron spraypositive; column: HP-Eclipse XDB-C18, 2×50 mm, buffer A:H₂O with 0.1%TFA, buffer B: MeCN with 0.1% TFA, elution gradient: 5-95% buffer over3.5 to 6 minute period, flow rate: 0.8 to 0.4 mL/min). Purity of thecompounds was also evaluated by HPLC using a Waters Millenniumchromatography system with a 2690 Separation Module (Milford, Mass.).The analytical columns was Alltima C-18 reversed phase, 4.6×250 mm fromAlltech (Deerfield, Ill.). A gradient elution was used, typicallystarting with 5% MeCN/95% water and progressing to 100% MeCN over aperiod of 40 minutes. All solvents contained 0.1% TFA. Compounds weredetected by ultraviolet light (UV) absorption at 214 mm. Some of themass spectrometric analysis was performed on a Fisons Electrospary MassSpectrometer. All masses are reported as those of the protonated parentions unless otherwise noted. Nuclear magnetic resonance (NMR) analysiswas performed with a Varian 300 MHz NMR (Palo Alto, Calif.). Thespectral reference was either TMS or the known chemical shift of thesolvent. Proton NMR (¹H NMR) data are reported as follows: chemicalshift (δ) in ppm, multiplicity (s=singlet, d=doublt, t=triplet,q=quartet, p=pentet, m=multiplet, dd=double of doublet, br=broad),coupling constant (Hz), integration and assignment. Melting points weredetermined on a Laboratory Devices MEL-TEMP apparatus (Holliston, Mass.)and are reported uncorrected.

Compound names were generated using NOMENCLATOR (ChemInnovationSoftware, Inc. San Diego, Calif.)

5.1.2 Synthesis of Estrogen Receptor-Modulating Isoxazoles

5.1.2.1 Synthesis of4-[5-[2-(4-hydroxyphenyl)ethyl]-4-benzylisoxazol-3-yl]phenol

This compound was synthesized by following Scheme 1 and Scheme 3 using4′-methoxyacetophenone and methyl-3-(4-methoxyphenyl)propanoate for theClaisen condensation and benzyl bromide for the alkylation.Demethylation was performed using Method 3 described in Step C of 1.ESMS m/z 372 (MH⁺), C₂₄H₂₁NO₃=371 g/mol; HPLC purity=70%.

5.1.2.2 Synthesis of 4-[4-ethyl-5-(phenoxymethyl)isoxazol-3-yl]phenol

The compound was synthesized by following the methods described abovefor Scheme 1 using 1-(4-methoxyphenyl)butan-1-one and 2-phenoxyacetylchloride as starting materials. Demethylation was performed using Method3 described for Step C of Scheme 1. ESMS m/z 296 (MH⁺), C₁₈H₁₇NO₃=295g/mol; HPLC purity=60%.

5.1.2.3 Synthesis of 4-[5-(4-hydroxyphenyl)-4-phenylisoxazol-3-yl]phenol

This compound was synthesized by following methods described above forScheme 1 using 1-(4-methoxyphenyl)-2-phenylethan-1-one and4-methoxybenzoyl chloride as starting materials. Demethylation wasperformed using Method 1 described for Step C in Scheme 1.

¹H NMR (D₆-DMSO) δ7.60-7.36 (m, 9H), 7.00-6.90 (m, 4H); MS m/z 330(MH+), C₂₁H₁₅NO₃=329 g/ml; HPLC purity=80%.

5.1.2.4 Synthesis of 4-[4-ethyl-5(4-hydroxyphenyl)isoxazol-3-yl]phenol

This compound was synthesized by following methods described above theScheme 1 using 1-(4-methoxyphenyl)-butane-1-one and 4-methoxybenzoylchloride as starting materials. Demethylation was performed using Method1 described for Step C in Scheme 1.

¹H NMR (d₆-DMSO) δ7.70 (d, J=8.24 Hz, 2H,) 7.50 (d, J=8.24 Hz, 2H), 7.00(dd, J=2.4, 2.2 Hz, 4H), 2.80 (q, J=6.75 Hz, 2H), 1.20 (t, J=6.75 Hz,3H); GCMS m/z 281 (M+), C₁₇H₁₅NO₃=281 g/mol; HPLC purity=90%.

5.1.2.5 Synthesis of4-[5-[2-(4-hydroxyphenyl)ethyl]-4-phenylisoxazol-3-yl]phenyl

This compound was synthesized by following methods described above forScheme 1 using 1-(4-methoxyphenyl)-2-phenylethan-1-one and 4-nitrophenyl3-(4-methoxyphenyl)propanoate as starting materials. Demethylation wasperformed using Method 3 described for Step C in Scheme 1.

ESMS m/z 358 (MH⁺), C₂₃H₁₉NO₃=357 g/mol; HPLC purity=70%

5.1.2.6 Synthesis of 4-[5-(4-hydroxyphenyl)-4-benzylisoxazol-3-yl]phenol

This compound was synthesized by following methods described above forScheme 1 using 1-(4-methoxyphenyl)-3-phenylpropan-1-one and p-anisoylchloride as starting materials. Demethylation was performed using Method1 described for Step C in Scheme 1.

¹H NMR (d₄-MeOH) δ7.60-7.20 (m, 9H), 7.01-6.80 (m, 4H), 4.20 (s, 2H); MSm/z 344 (MH+), C₂₂H₁₇NO₃=343 g/mol; HPLC purity=80%.

5.1.2.7 Synthesis of 4-[3.5-bis(4-hydroxyphenyl)isoxazol-4-yl]phenol

This compound was synthesized by following Scheme 1 and Scheme 3.

Step 1: To a solution of 4′-methoxyacetophenone in THF at −78° C. wasadded dropwise 1.5 eq. of [(CH₃)₂Si]₂NLi. The solution was stirred for 1h at −78° C., followed by addition of 1.2 eq. of p-anisoyl chloride. Thereaction mixture was stirred for 10 min at −78° C. and then for 22 h atrt, acidified with 10% citric acid, and extracted with BtOAc. Thecombined organic layers were washed with water and dried over Na₂SO₄.Removal of solvent in vacuo provided a crude solid which was purified byflash chromatography (CH₂Cl₂) to give 1,3-diketone as a white solid.

Step 2: Synthesis of p-methoxyphenyllead triacetone. To a solution oflead tetraacetate (0.73 equiv) in chloroform and dichloroacetic acid wasadded anisole (1.0 equiv.). The reaction mixture was allowed to stir atroom temperature for 90 min. After this period the solution was washedwith water and the organic layer was treated with hexane. The productprecipitated out and was collected by filtration. The solid was taken upin chloroform and acetic acid. The resultant solution was stirred for 1h and then washed with water. The later 2 steps were repeated and thechloroform layer was treated with hexane. This mixture was cooled to 2°C. for 24 h. The material that precipitated was collected by filtrationand dried under vacuum to afford p-methoxyphenyllead triacetate.

Step 3: A mixture of the 1,3-diketone (1.0 equiv., obtained from 1),p-methoxyphenyllead triacetate (1.1 equiv. obtained from step 2) andpyridine (3.3 equiv.) in chloroform was stirred at room temperature for48 h. After this time had elapsed the reaction was diluted withchloroform, washed with water and 4M sulfuric acid. The aqueous washeswere back extracted with chloroform. The combined organic extracts werewashed with water, dried over magnesium sulfate, filtered andconcentrated in vacuo. The crude product was purified by flash columnchromatography (ethyl acetate/hexane, 1:3) to afford an off white solidas 1,2,3-tri(4-methoxyphenyl)propane-1,3-dione.

Step 4: A mixture of the 1,3-diketone obtained in step 3 (1.0 equiv.),hydroxylamine hydrochloride (1.5 equiv.), pyridine (2.0 equiv.) andethanol was heated to reflux overnight. Cooled in rt and removed solventin vacuo. Water and ethyl acetate were added. The organic layer wasseparated, washed with dil. HCl, lurine, dried, filtered and the solventwas concentrated in vacuo. The product1-[4,5-bis(4-methoxyphenyl)isoxazol-3-yl]-4-methoxybenzene was obtainedas an off while solid.

Step 5: Dimethylation was performed using Method 1 described for Step Cin Scheme 1 to afford the product4-[3,5-bis(4-hydroxphenyl)isoxazol-4-yl]phenol.

¹H NMR (d₆-DMSO) δ7.30 (d, J=9.0 Hz, 2H), 7.20 (d, J=9.0 Hz, 2H), 7.10(d, J=9.0 Hz, 2H), 6.80 (d, J=9.0 Hz, 2H), 6.76 (d, J=9.0 Hz, 2H), 6.74(d, J=9.0 Hz, 2H); MS m/z 346 (MH+), C₂₁H₁₅NO₄=345 g/mol; HPLCpurity=97%.

5.1.2.8 Synthesis of 4-[5-(4-hydroxphenyl)isoxazol-3-yl]phenol

This compound was synthesized by following the methods described abovefor Scheme 1 using 4′-acetophenone and p-anisoyl chloride as startingmaterials. Demethylation was performed using Method 1 described for StepC in Scheme 1.

¹H NMR (d₆-DMSO) δ7.80-7.75 (m, 4H), 7.5 (s, 1H), 6.99-6.90 (m, 4H); MSm/z 254 (MH+), C₁₅H₁₁NO₃=253 g/mol; HPLC purity=96%.

5.1.2.9 Synthesis of4-[5-(4-hydroxyphenyl)-4-[4-(2-piperidylethoxy)phenyl]isoxazol-3-yl]phenol

This compound was synthesized by following Scheme 1, Scheme 3, Scheme 5.

Step 1: Synthesis of 1,3-diketone is the same as Step 1 of the Examplein Section 5.2.2.7.

Step 2: Synthesis of p-allyloxyphenyllead triacetate. Similar as Step 2in Section 5.1.2.7 except alkylphenyl ether was used as the startingmaterial instead of p-anisole.

Step 3: Similar as Step 3 in Section 5.1.2.7, usingp-allyloxyphenylphenyllead triacetate instead of p-methoxyphenylleadtriacetate.

Step 4: Formation of isoxazole skeleton is the same as Step 4 of Section5.1.2.7. The product obtained from this step is1-[3,5-bis(4-methoxyphenyl)isoxazol-4-yl]-4-prop-2-enyloxybenzene.

Step 5: Selective removal of the allyl protecting group. A mixture ofthe above isoxazole (1.0 equiv.), pyrrolidine (20 equiv.),triphenylphosphine (0.05 equiv.) and tetrakis(tiphenylphosphine)palladium(0) (0.05 equiv.) in THF was heated to reflux overnight. Thesolution was concentrated in vacuo and the crude material was purifiedby flash column chromatography (ethyl acetate/hexane 1:3). The product4-[3,5-bis(4-methoxyphenyl)isoxazol-4-yl]phenol was obtained as a whitesolid.

Step 6: A mixture of the phenol (1.0 equiv., obtained from step 5),1-(2-chloroethyl)piperidine monohydrochloride (1.2 equiv.) cesliumcarbonate (2.5 equiv.) in DMF (30 ml) was heated at 100° C. overnight.The solids were removed by filtration, the filtrate was concentrated invacuo and the residue was taken up into ethyl acetate. The solution waswashed with water, brine, dried over sodium sulfate, filtered andconcentrated in vacuo to give1-[3,5-bis(4-methoxyphenyl)isoxazol-4-yl]-4-(2-piperidylethoxy)benzene.

Step 7: Dimethylation was performed using Method 2 described for Step Cin Scheme I. To a solution of the isoxazole obtained from step 6 (1.0equiv.) in dichloroethane was added aluminum chloride (5.0 equiv.) andethane thiol (5.0 equiv.). The resultant mixture was stirred for 40 minat room temperature and quenched with THF, 20% HCl and water. Aprecipitate was formed and this was collected by filtration. Afterdrying the product the material was taken up in methanol, treated withactivated charcoal, filtered through sodium sulfate and concentrated invacuo. Purification with flash chromatography followed by addition ofHCl aq. and lyophilization, then afforded the product4-[5-(4-hydroxyphenyl)-4-[4-(2-piperidylethoxy)phenyl]isoxazol-3-yl]phenolas hydrochloride salt.

¹H NMR (d₄-MeOH) δ7.43 (d, J=9.0Hz, 2H), 7.25 (d, J=8.0Hz, 2H), 7.20 (d,J=9.0Hz, 2H), 7.03 (d, J=9.0Hz, 2H), 6.77 (d, J=8.0Hz, 2H), 6.75 (d,J=8.0Hz, 2H), 4.20 (t, J=5.5Hz, 2H), 2.90 (t, J=5.5Hz, 2H), 2.60-2.70(m, 4H), 1.63-1.75 (m, 4H), 1.50-1.60 (m, 2H); MS m/z 457 (MH⁺, 100%)C₂₈H₂₈N₂O₄=456 g/mol; HPLC purity=98%.

5.1.2.10 Synthesis of4-[4-(4-hydroxphenyl)-3-[4-(2-piperidylethoxy)phenyl]isoxazol-5-yl]phenol

This compound was synthesized by following the procedures describedabove in Scheme 1, Scheme 3, and Scheme 7.

Step 1: Synthesis of 1,3-diketone is similar to that described in Step 1of Section 5.1.2.7. To a solution of desoxyanisoin (1.0 equiv) in THF at−78° C. was added lithium bis(trimethylsilyl)amide (1.2 equiv.)dropwise. The resultant solution was allowed to stir for 40 min at −78°C. and a solution of p-allyloxybenzoyl chloride (1.1 equiv.) in THF wasadded dropwise. The reaction mixture was allowed to warm to roomtemperature slowly overnight to produce an orange colored solution. Themixture was diluted with 0.5N HCl, extracted with ethyl acetate, washedwith 0.5N HCl, water, brine, dried over sodium sulfate, filtered andconcentrated in vacuo. The crude product was purified byrecrystallization. This afforded some of the desired compound and theremaining residue was purified by flash column chromatography (ethylacetate:hexanes 1:3) to afford the 1,3-diketone.

Step 2: Formation of isoxazole is the same as Step 4of Section 5.1.2.7.The product afforded from this step is4-methoxy-1-[4-(4-methoxyphenyl)-5-(4-prop-2-enyloxyphenyl)isoxazol-3-yl]benzene.

Step 3: Selective removal of the allyl protecting group. Same as Step 5of Section 5.1.2.9. The product afforded from this step is4-[3,4-bis(4-methoxyphenyl)isoxazol-5-yl]phenol.

Step 4: Alkylation. Same as Step 6 Section 5.1.2.9. The product affordedfrom this step is4-methoxy-1-{4-(4-methoxyphenyl)-5-[4-(2-piperidylethoxy)phenyl]isoxazol-3-yl}benzene.

Step 5: Demethylation was performed using Method 2 described for Step Cis Scheme 1 (see Step 7 of Section 5.1.2.9). The product was4-{4-(4-hydroxyphenyl)-3-[4-(2-piperidylethoxy)phenyl]isoxazole-5-yl}phenol.

¹H NMR (d₆-DMSO) δ6.70-7.50 (m, 2H), 4.35-4.42 (m, 2H), 3.30-3.50 (m,2H), 2.90-3.25 (m, 4H), 1.60-1.80 (m, 4H), 1.30-1.45 (m, 2H); MS m/z 457(MH⁺, 100%), C₂₈H₂₈N₂O₄=456 g/mol; HPLC purity=98%.

5.1.2.11 Synthesis o f3-[4,5-bis(4-hydroxyphenyl)isoxazol-3-yl]phenol

This compound was synthesized by following Scheme 1. Desoxyanisoin andm-anisoyl chloride were used as starting materials. Demethylation wasperformed using Method 1 described for Step C in Scheme 1. The productobtained was an off-white solid.

MS m/z 346 (MH+), C₂₁H₁₅NO₄=345 g/mol; HPLC purity=95%.

5.1.2.12 Synthesis of 4-[4,5-bis(4-hydroxyphenyl)isoxazol-3-yl]phenol

This compound was synthesized by following Scheme 1. Desoxyanisoin ando-anisoyl chloride were used as starting materials. Demethylation wasperformed using Method 1 described for Step C in Scheme 1. The productobtained was an off-white solid.

MS m/z 346 (MH+), C₂₁H₁₅NO₄=345 g/mol; HPLC purity=96%

5.1.2.13 Synthesis of4-[3-(4-hydroxyphenyl)-4-methylisoxazol-5-yl]phenol

This compound was synthesized by following Scheme 1,1-(4-methoxyphenyl)propan-1-one and p-anisoyl chloride were used asstarting materials. Demethylation was performed using Method 1 describedfor Step C in Scheme 1.

ESMS m/z 268 (MH⁺), C₁₆H₁₃NO₃=267 g/mol; HPLC purity=85%.

5.1.2.14 Synthesis of4-[3-(4-hydroxyphenyl)-5-phenylisoxazol-4-yl]phenol

This compound was synthesized by following Scheme 1. Desoxyanisoin andbenzoyl chloride were used as starting materials. Demethylation wasperformed using Method 1 described for Step C in Scheme 1.

ESMS m/z 330 (MH⁺), C₂₁H₁₅NO₃=329 g/mol; HPLC purity=90%

5.1.2.15 Synthesis of4-[5-(4-fluorophenyl)-3-(4-hydroxphenyl)isoxazol-4-yl]phenol

This compound was synthesized by following Scheme 1. Desoxyanisoin andp-fluorobenzoyl chloride were used as starting materials. Demethylationwas performed using Method 1 described for Step C in Scheme 1.

ESMS m/z 348 (MH⁺), C₂₁H₁₄FNO₃=347 g/mol, HPLC purity=85%

5.1.2.16 Synthesis of4-[4-(4-hydroxyphenyl)-5-[3-(trifluoromethoy)phenyl]isoxazol-3-yl]phenol

This compound was synthesized by following Scheme 1. Desoxyanisolin andm-trifluoromethylbenzoyl chloride were used as starting materials.Demethylation was performed using Method 1 described for C in Scheme 1.

ESMS m/z 414 (MH²³H₁₄F₃NO_(b)=413 g/mol; HPLC purity=90%

5.1.2.17 Synthesis of4-[4-(4-hydroxphenyl)-5-[4-(trifluoromethoxy)phenyl]isoxazol-3-yl]phenol

This compound was synthesized by following Scheme 1. Desoxyanisoin andp-trifluoromethylbenzoyl chloride were used as starting materials.Demethylation was performed using Method 1 described for Step C isScheme 1.

ESMS m/z 414 (MH⁺), C₂₂H₁₄F₃NO₄=413 g/mol; HPLC purity=90%

5.1.2.18 Synthesis of4-[5-(4-hydroxphenyl)-4-(phenoxymethyl)isoxazol-3-yl]phenol

This compound was synthesized by following the procedures described forScheme 1 and Scheme 8.

Step 1: Formation of 1,3-diketone, Same as Step 1 of Section 5.1.2.7,except 1-(4-methoxyphenyl)propan-1-one was used as a starting material.

Step 2: Formation of the isoxazole skeleton. Same as Step 4 of Section5.1.2.7. The product obtained from this step was4-methoxy-1-[5-(4-methoxyphenyl)-4-methylisoxazol-3-yl]benzene.

Step 3: Bromination of 4-methylisoxazole. A suspension of the above4-methylisoxazole (1.0 eq.), N-bromosuccinimide (NBS), (1.1 eq.), andPhCO₃H (catalytic amount) in CCl₄ was heated to reflux under argon for 2h, cooled to rt and filtered. The filtrate was diluted with DCM, washedwith 10% Na₂S₂O₃ and 10% NaHCO₃, dried and concentrated in vacuo. Theresulting crude material was purified with flash column chromatographyto give product1-[4-(bromomethyl)-5-(4-methoxyphenyl)isoxazol-3-yl]-4-methoxybenzene.

Step 4: Alkylation. To a solution of phenol (1.1 eq.) in dry THF at 0°C. was added powder NaH (1.1 eq.) under argon. When the solution stoppedbubbling, the above bromide was added to the solution and the mixturewas stirred overnight at rt. The resulting solution was acidified withsaturated NH₄Cl and extracted with ethyl ether. The combined organiclayers were dried over Na₂SO₄ and rotary evaporated to give a whitesolid.

Step 5: Demethylation. The above white solid was converted to product4-[5-(4-hydroxyphenyl)-4-(phenoxymethyl)isoxazol-3-yl]phenol usingMethod 1 described for Step C of Scheme 1.

ESMS m/e 360 (MH⁺), C₂₂H₁₇NO₄=359 g/mol; HPLC purity=90%.

5.1.2.19 Synthesis of4-[5-(4-hydroxphenyl)-4(phenythiomethyl)isoxazol-3-yl]phenol

This compound was synthesized by following the procedures described forScheme 1 and Scheme 8.

Step 1-3 are the same as described for Steps 1-3 of Section 5.1.2.18,respectively.

Step 4: Alkylation. Same as Step 4 of section 5.1.2.18, exceptthiophenol was used instanced of phenol.

Step 5: Demethylation. Same as Step 5 of Section 5.1.2.18. The productobtained was4-[5-(4-hydroxphenyl)-4-(phenylthiomethyl)isoxazol-3-yl]phenol.

ESMS m/e 376 (MH⁺), C₂₂H₁₇NO₃S=375 g/mol; HPLC purity=90%

5.1.2.20 Synthesis of3,5-bis(4-hydroxphenyl)isoxazol-4-yl-4-(2-piperidylethoxy)phenyl ketone

This compound was synthesized by following the procedures describedabove for Scheme 5.

Step 1: Same as Step 1 described in Section 5.1.2.7.

Step 2: Formation of isoxazole heterocycle. Same as Step 4 in Section5.1.2.7. The product obtained from this step was4-methoxy-1-[5-(4-methoxyphenyl)isoxazol-3-yl]benzene.

Step 3: Bromination. To a solution of the above isoxazole (1.0 ea.) inanhydrous CHCl₃ at reflux (70° C.) under argon was added dropwisebromine (1.01 eq.) in anhydrous CHCl₃ solution. The mixture was stirredfor 50 min at reflux, followed by addition of 10% Na₂S₂O₃ in saturatedNaHCO₃ aqueous solution. The aqueous-organic solution was separated andthe aqueous layer was extracted with CH₂Cl₂. The combined organic phaseswere dried over Na₂SO₄ and rotary evaporated to give a yellow solid thatwas washed with ethyl acetate (“EtOAc”) to afford pale yellow solid as1-[4-bromo-5-(4-methoxyphenyl)isoxazol-3-yl]-4-methoxybenzene in 92.5%yield.

Step 4: Acylation. To a solution of the above 4-bromoisoxazole (1.0 eq.)in anhydrous THF at −98° C. under argon was added n-BuLi (1.2 eq., 1.6 Min hexane). The resultant solution was stirred for 1 h at −98° C. andthen transferred dropwise to a solution of 4-allyoxybenzoyl chloride(1.2 eq.) in THF at −78° C. through a double-tipped needle. The reactionmixture was stirred overnight at −78° C., diluted with water, and thenacidified with 10% aqueous citric acid. The organic layer was dried overNa₂SO₄ and purified with flash chromatography (CH₂Cl₂) to give acylatedproduct 3,5-bis(4-methoxyphenyl)isoxazol-4-yl 4-prop-2-enyloxyphenylketone as a pale yellow solid in 64% yield.

Step 5: Selective removal of the allyl protecting group. Same as Step 5of Section 5.1.2.9.

Step 6: Alkylation. Same as Step 6 of Section 5.1.2.9.

Step 7: Demethylation was following Method 2 described for Step C ofScheme 1. To a solution of the compound obtained from the above step 6in dry CH₂Cl₂ was added 5.0 equivalent of AlCl₃ and 5.0 equivalents ofethane thiol (“EtSH”). The reaction mixture was stirred at rt for 1.2 hand the resultant slurry was then poured to ice-water. The organic layerwas separated. The aqueous layer was extracted with EtOAc three times.The combined organic layers were dried over Na₂SO₄ and rotary evaporatedin vacuo. The obtained reaction mixture which contained desired productand side product as mono-demethylated compound. The mixture wasseparated by HPLC to give product 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-(2-piperidylethoxy)phenyl ketone in 22% yield after HPLC purification.

The product obtained from HPLC purification was dissolved in EtOAc/ sat.NaHCO₃ (1:1) solution. The bilayer solution was shaken vigorously andseparated. The aqueous layer was extracted with EtOAc several times. Thecombined organic layer was dried over MgSO₄ and rotary evaporated togive a yellow solid. The solid was then dissolved in cold acetone/conc.HCl (2:1) and the solution was rotary evaporated in vacuo to produce theHCl salt of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-(2-piperidylethoxy)phenyl ketone as an off white powder.Recrystallization with methanol/water gave the salt as fine flakycrystals.

¹H NMR (d₆-DMSO) δ10.2 (s, 1H, OH), 9.93 (s, 1H, OH), 7.8-6.72 (m, 12H,3Ph), 4.4 (brt, 2H, OCH₂-), 3.49 (m, 4H, N(CH₂)₂), 2.98 (brt, 2H,—CH₂-N), 1.79-1.39 (m, 6H) —(CH₂)₃). ESMS m/e 485 (MH⁺), C₂₉H₂₈N₂O₅=484g/mol; HPLC purity=99% mp 255° C. decomposed.

5.1.2.21 Synthesis of5-(4-hydroxyphenyl)-3-(4-methoxyphenyl)isoxazol-4-yl4-(2-piperidhylethoxyphenyl ketone

This compound was synthesized by following Scheme 5.

Steps 1-6 are exactly the same as the corresponding Steps of Section5.1.2.20.

Step 7: Same as Step 7 of Section 5.1.2.20. Partial demethylation gavethe title compound 5-(4-hydroxphenyl)-3-(4-methoxyphenyl)isoxazol-4-yl4-(2-piperidylethoxy)phenyl ketone which was isolated by HPLC.

ESMS m/e 499 (MH⁺), C₃₀H₃₀N₂O₅=498 g/mol; HPLC purity=97%.

5.1.2.22 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-[2-(hydroxypiperidyl)ethoxy]phenyl ketone

This compound was obtained as a side product of the reactions describedin Section 5.1.2.20. The crude material obtained from Step 7 of Section5.1.2.20 was dissolved in DMSO and left at rt overnight. HPLC analysisof this mixture showed formation of new compound that was 16 mass unitshigher than any of the products found in the reaction of Step 7 ofSection 5.1.2.20. HPLC isolation thus afforded N-oxide3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-[2-hydroxypiperidyl)ethoxy]phenyl ketone.

¹H NMR (d₆-DMSO): δ 10.22 (s, 1H), 9.94 (s, 1H), 7.82-7.79 (d, J=8.76Hz, 2H), 7.46-7.43 (d, J=8.76 Hz, 2H), 7.34-7.32 (d, J=8.30 Hz),7.04-7.01 (d, J=8.76 Hz, 2H), 6.83-6.80 (d, J=8.76 Hz, 2H), 6.78-6.75(d, J=8.30 Hz, 2H), 4.55 (br, 2H), 4.05 (br, 2H), 3.67 (br, 4H),2.00-1.35 (m, 6H); LC/MS m/z 501 (M⁺), C₂₉H₂₈N₂O₆=500 g/mol; HPLCpurity=99%.

5.1.2.23 Synthesis of3-(4-hydroxyphenyl)-5-(4-methoxyphenyl)isoxazol-4-yl4-[2-(hydroxypiperidyl)ethoxy]phenyl ketone

This compound was obtained as a side product of the synthesis describedin Section 5.1.2.21. The crude material obtained from Step 7 wasdissolved in DMSO and left at rt overnight. HPLC analysis of thismixture showed formation of new compound which was 16 mass units higherthan any of the products characterized previously. HPLC isolationafforded 3-(4-hydroxyphenyl)-5-(4-methoxyphenyl)isoxazol-4-yl4-[2-(hydroxypiperidyl)ethoxy]phenyl ketone.

ESMS m/e 515 (MH⁺), C₃₀H₃₀N₂O₆=514 g/mol); HPLC purity=88%.

5.1.2.24 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-hydroxyphenyl ketone

This compound was synthesized by following the procedures described forScheme 5.

Steps 1-4 were performed exactly the same as corresponding Steps inSection 5.1.2.20.

Step 5: Deprotection was performed by following Method 3 described forStep C in Scheme 1. This step gave the desired product3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 4-hydroxyphenyl ketone.

ESMS m/e 374 (MH⁺), C₂₂H₁₅NO₅=373 g/mol; HPLC purity=90%.

5.1.2.25 Synthesis of 4-[4-bromo-5-(4-hydroxyphenyl)isoxazol-3-yl]phenol

This compound was synthesized by following the procedure described forScheme 4.

Step 1-3 are exactly the same as the corresponding steps in Section5.1.2.20.

Step 4: Demethylation was performed using Method 1 described for Step Cin Scheme 1.

ESMS m/e 332/334 (MH⁺), C₁₅H₁₀BrNO₃=331/333 g/mol (1 Br); HPLCpurity=90%.

5.1.2.26 Synthesis of4-[4-(bromomethyl)-5-(4-hydroxyphenyl)isoxazol-3-yl]phenol

This compound was synthesized by following the procedures describedabove for Scheme 1 and Scheme 8.

Steps 1-3 are same as corresponding steps in Section 5.1.2.20.

Step 4: Demethylation was following Method 3 described for Step C inScheme 1. To a solution of above isoxazole in DCM at −78 C was addeddropwise 5 eq. boron tribromide (1M in DCM solution). The mixture wasslowly warmed to it and stirred under argon for 1.5 h. The reaction wasquenched by adding water and neutralized to pH 5. Extracted with EtOAc,dried with Na₂SO₄ and concentrated in vacuo to give a crude material.Purification by HPLC afforded 10% of desired product.

ESMS m/e 346/348 (MH⁺, 100%), C₁₆H₁₂BrNO₅=345/347 g/mol (1 Br); HPLCpurity=80%.

5.1.2.27 Synthesis of4-{5-(4-hydroxyphenyl)-4-[(4-hydroxyphenoxy)methyl]isoxazol-3-yl}phenol

This compound was synthesized by following the procedures describedabove for Scheme 1 and Scheme 8.

Steps 1, 2 and 3 are same as corresponding steps in Section 5.1.2.18.

Step 4: Alkylation. Same as step 4 of Section 5.1.2.18 except4-(benzyloxy)phenol was used in the procedure. The product obtained formthis step is1-{[3,5-bis(4-methoxyphenyl)isoxazol-4-yl]methoxy}-4-(phenylmethoxy)benzene.

Step 5: Demethylation was following Method 3 described for Step C inScheme 1. The product obtained was4-{5-(4-hydroxyphenyl)-4-[4-hydroxyphenoxy)methyl]isoxazol-3-yl}phenol.

ESMS m/e 376 (MH⁺), C₂₂H₁₇NO₄=375 g/mol; HPLC purity=80%.

5.1.2.28 Synthesis of4-[4-(hydroxymethyl)-5-(4-hydroxyphenyl)isoxazol-3-yl]phenol

This compound was obtained as a side product in the synthesis of productin Section 5.1.2.27; isolation was achieved with HPLC.

ESMS m/z 284 (MH⁺), C₁₆H₁₃NO₄=283 g/mol, HPLC purity=99%.

5.1.2.29 Synthesis of3-(4-hydroxyphenyl)-4,5-dihydronaphtho[1,2-c]isoxazol-7-ol

This compound was synthesized by following the methods described inScheme 1, using 6-methoxy-1-tetralone and p-anisoyl chloride as thestarting materials. Demethylation was performed following Method 1 ofStep C in Scheme 1.

ESMS m/e 280 (MH⁺), C₁₇H₁₃NO₃=279 g/mol; HPLC purity=90%.

5.1.2.30 Synthesis of4-(7-methoxy-4,5-dihydronaphtho[1,2-c]isoxazol-3-yl)phenol

This compound was synthesized in the same manner as described in Section5.1.2.29. Partial demethylation afforded mono-methylated product4-(7-methoxy-4,5-dihydronaphtho[1,2-c]isoxazol-3-yl)phenol, which wasisolated by HPLC to provide the desired product.

ESMS m/e 294 (MH⁺), C₁₈H₁₅NO₃=293 g/mol; HPLC purity=89%.

5.1.2.31 Synthesis of 3-[3-(4-hydroxyphenyl)isoxazol-5-yl[phenol

This compound was synthesized in the same manner as described in Section5.1.2.8 except 4′-methoxyacetophenone and m-anisoyl chloride were usedas the starting materials.

ESMS m/e 254 (MH⁺), C₁₅H₁₁NO₃=253 g/mol; HPLC purity=90%.

5.1.2.32 Synthesis of 3-(4-hydroxyphenyl)naphtho[1,2-c]isoxazol-7-ol

This compound was synthesized by following the methods described inScheme 1.

Step 1: Formation of 1,3-diketone. Same as Step 1 of Section 5.1.2.7except 6-methoxy-1-tetralone and p-anisoyl chloride were used as thestarting materials.

Step 2: Formation of isoxazole heterocycle. Same as Step 4 of Section5.1.2.7. The product obtained from this step was7-methoxy-3-(4-methoxyphenyl)-4,5-dihydronaphtho[1,2-c]isoxazole.

Step 3: Oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone(“DDQ”). To the product of Step 2 in dry toluene was added DDQ (1.1eq.). The solution was refluxed overnight, quenched with sat. NaHCO₃,K₂CO₃ and extracted with CH₂Cl₂. The combined organic layers were driedover Mg₂SO₄ and evaporated in vacuo to give a solid residue.Purification with flash column chromatography yield product7-methoxy-3-(4-methoxyphenyl)naphthio[1,2-c]isoxazole.

Step 4: Demethylation was following Method 1 described for Step C inScheme 1.

ESMS m/e 278 (MH⁺), C₁₇H₁₁NO₃=277 g/mol; HPLC purity=95%.

5.1.2.33 Synthesis of3-(3-hydroxyphenyl)4,5-dihydronaphtho[1,2-c]isoxazol-7-ol

This compound was synthesized in the same manner as described in Section5.1.2.29 except m-anisoyl chloride was used as one of the startingmaterials.

ESMS m/e 280 (MH⁺), C₁₇H₁₃NO₃=279 g/mol; HPLC purity=90%.

5.1.2.34 Synthesis of3-(2-hydroxyphenyl)-4,5-dihydronaphtho[1,2-c]isoxazol-7-ol

This compound was synthesized in the same manner as described in Section5.1.2.29, except o-anisoyl chloride was used as one of the startingmaterials.

ESMS m/e 280 (MH⁺), C₁₇H₁₃NO₃=279 g/mol; HPLC purity=90%.

5.1.2.35 Synthesis of 2-[5-(3-hydroxyphenyl)isoxazol-3-yl]phenol

This compound was synthesized in the same manner as described in Section5.1.2.8, except 3′-methoxyacetophenone and m-anisoyl chloride were usedas starting materials.

ESMS m/e 254 (MH⁺), C₁₅H₁₁NO₃=253 g/mol; HPLC purity=97%.

5.1.2.36 Synthesis of 2-[3-(3-hydroxyphenyl)isoxazol-5-yl]phenol

This compound was synthesized in the same manner as described in Section5.1.2.8, except 3′-methoxyacetophenone and o-anisoyl chloride were usedas starting materials.

ESMS m/e 254 (MH⁺), C₁₅H₁₁NO₃=253 g/mol; HPLC purity=96%.

5.1.2.37 Synthesis of 2-[5-(2-hydroxyphenol)isoxazol-3-yl]phenol

This compound was synthesized in the same manner as described in Section5.1.2.8 except 2′-methoxyacetophenone and o-anisoyl chloride were usedas starting materials.

ESMS m/e 254 (MH⁺), C₁₅H₁₁NO₃=253 g/mol; HPLC purity=90%.

5.1.2.38 Synthesis of3-[3-(3-hydroxyphenyl)-4-methylisoxazol-5-yl]phenol

This compound was synthesized by following the procedures described forScheme 1 and Scheme 3.

Step 1: Formation of 1,3-diketone is the same as Step 1 as described inSection 5.1.2.7 using 3′-methoxyacetophenone and m-anisoyl chloride asthe starting materials.

Step 2: Alkylation. A THF solution of the above 1,3-diketone (1.0 eq.)was added dropwise to a suspension of sodium hydride (1.1 eq) in THF at0° C. The mixture was stirred at rt for 30 min. followed by addition ofiodomethane (1.1 eq.). The reaction mixture was stirred at rt overnight,poured into saturated NH₄Cl aq. and extracted with ether and DCM. Theorganic extracts were washed with brine, dried with MgSO₄ andconcentrated in vacuo to give the product1,3-bis(3-methoxyphenyl)-2-methylpropane-1,3-dione.

Step 3: Formation of isoxazole heterocycle. Same as Step 4 as describedin Section 5.1.2.7.. The product obtained from this step is3-methoxy-1-[5-(3-methoxyphenyl)-4-methylisoxazol-3-yl]benzene.

Step 4: Demethylation was performed following Method 1 of Step C ofScheme 1 to give the desired product.

ESMS m/e 268 (MH⁺), C₁₆H₁₃NO₃=267 g/mol; HPLC purity=94%.

5.1.2.39 Synthesis of2-[3-(3-hydroxyphenyl)-4-methylisoxazol-5-yl]phenol

This compound was synthesized in the same manner as described in Section5.1.2.38, except 3′-methoxyacetophenone and o-anisoyl chloride were usedas starting materials.

ESMS m/e 268 (MH⁺), C₁₆H₁₃NO₃=267 g/mol; HPLC purity=90%.

5.1.2.40 Synthesis of2-[3-(2-hydroxyphenyl)-4-methylisoxazol-5-yl]phenol

This compound was synthesized in the same manner as described in Section5.1.2.38, except 2′-methoxyacetophenone and o-anisoyl chloride were usedas starting materials.

ESMS m/e 268 (MH⁺), C₁₆H₁₃NO₃=267 g/mol; HPLC purity=90%.

5.1.2.41 Synthesis of 3-[4-ethyl-3-(3-hydroxyphenyl)isoxazol-5-yl]phenol

This compound was synthesized by following the procedures described forScheme 1 and Scheme 3.

Steps 1 was performed as the corresponding Step described in Section5.1.2.38.

Step 2 alkylation procedure was performed as Step 2 of Section 5.1.2.38,using iodoethane as the alkylating agent.

Steps 3 and 4 are exactly the same as the corresponding Steps describedin Section 5.1.2.38. The product obtained was3-[4-ethyl-3-(3-hydroxyphenyl)isoxazol-5-yl]phenol.

ESMS m/e 282 (MH⁺), C₁₇H₁₅NO₃=281 g/mol; HPLC purity=90%.

5.1.2.42 Synthesis of 2-[4-ethyl-3-(3-hydroxyphenyl)isoxazol-5-yl]phenol

This compound was synthesized in the same manner described in Section5.1.2.41, except 3′-methoxyacetophenone and o-anisoyl chloride were usedas starting materials.

ESMS m/e 282 (MH⁺), C₁₇H₁₅NO₃=281 g/mol; HPLC purity=90%.

5.1.2.43 Synthesis of 2-[4-ethyl-3-(3-hydroxyphenyl)isoxazol-5-yl]phenol

This compound was synthesized in the same manner described in section5.1.2.41, except 2′-methoxyacetophenone and o-anisoyl chloride were usedas starting materials.

ESMS m/e 282 (MH⁺), C₁₇H₁₅NO₃=281 g/mol; HPLC purity=90%.

5.1.2.44 Synthesis of3-[4-(4-hydroxyphenyl)-5-(3-hydroxyphenyl)isoxazol-3-yl]phenol

This compound was synthesized in the same manner described in Section5.1.2.7, except 3′-methoxyacetophenone and m-anisoyl chloride were usedas starting materials.

ESMS m/z 346 (MH+), C₂₁H₁₅NO₄=345 g/mol; HPLC purity=94%.

5.1.2.45 Synthesis of2-[3-(3-hydroxyphenyl)-4-(4-hydroxyphenyl)isoxazol-5-yl]phenol

This compound was synthesized in the same manner described in Section5.1.2.7, except 3′-methoxyacetophenone and o-anisoyl chloride were usedas starting materials.

ESMS m/z 346 (MH+), C₂₁H₁₅NO₄=345 g/mol; HPLC purity=90%.

5.1.2.46 Synthesis of2-[4-(4-hydroxyphenyl)-5-(2-hydroxyphenyl)isoxazol-5-yl]phenol

This compound was synthesized in the same manner described in Section5.1.2.2, except 2′-methoxyacetophenone and o-anisoyl chloride were usedas starting materials.

ESMS m/z 346 (MH+), C₂₁H₁₅NO₄=345 g/mol; HPLC purity=94%.

5.1.2.47 Synthesis of3-(2-hydroxyphenyl)-4,5-dihydronaphtho[2,1-d]isoxazol-7-ol

This compound was synthesized in the same manner as described in Section5.1.2.29, except 6-methoxy-1-tetralone and o-anisoyl chloride were usedas starting materials. Demethylation was performed following Method 1described in Step C of Scheme 1 to provide the desired product.

ESMS m/e 280 (MH⁺), C₁₇H₁₃NO₃=279 g/mol; HPLC purity=90%.

5.1.2.48 Synthesis of2-(7-methoxy-4,5-dihydronaphtho[1,2-c]isoxazol-3-yl]phenol

This compound was synthesized in the same manner as described in Section5.1.2.47. Partial demethylation afforded mono-methylated product2-(7-methoxy-4,5-dihydronaphtho[1,2-c]isoxazol-3-yl)phenol.

ESMS m/e 294 (MH⁺), C₁₈H₁₅NO₃=293 g/mol; HPLC purity=95%.

5.1.2.49 Synthesis of2-[5-(3-hydroxyphenyl)-4-methylisoxazol-3-yl]phenol

This compound is the regioisomer of the compound described in Section5.1.2.39. Both regioisomers were obtained using the methods described inthat Section, and the two regioisomers were separated by HPLC using C₁₈column (Reliasil-BDXC18, 10×50 mm, Ranin Dynamax) running a first bufferof H₂O/0.1% TFA and a second buffer of HCN/0.1% TFA through a gradientfrom 5-95% of the second buffer over a nice-minute period at a flow rateof ten ml/min.

ESMS m/e 268 (MH⁺), C₁₆H₁₃NO₃=267 g/mol; HPLC purity=90%.

5.1.2.50 Synthesis of 2-[4-ethyl-5-(3-hydroxyphenyl)isoxazol-3-yl]phenol

This compound is the regioisomer of compound desired in Section5.1.2.42. Both regioisomers were obtained using the methods described inthat Section, and the two regioisomers were separated by HPLC using aC₁₈ column (Reliasil-BDXC18, 10×50 mm, Ranin Dynamax) running a firstbuffer of H₂O/0.1TFA and a second buffer of HCN/0.1% TFA through agradient from 5-95% of the second buffer over a nine-minute period at aflow rate of ten ml/min.

ESMS m/e 282 (MH⁺), C₁₇H₁₅NO₃=281 g/mol; HPLC purity=90%.

5.1.2.51 Synthesis of2-[4-(4-hydroxyphenyl)-5-(3-hydroxyphenyl)isoxazol-3-yl]phenol

This compound is the regioisomer of the compound described in Section5.1.2.45. Both regioisomers were obtained using the methods described inthat Section, and the two regioisomers were separated by HPLC using aC₁₈ column (Reliasil-BDXC18, 10×50 mm, Ranin Dynamax) running a firstbuffer of H₂O/0.1% TFA and a second buffer of HCN/0.1% TFA through agradient from 5-95% of the second buffer over a nine-minute period at aflow rate of ten ml/min.

ESMS m/z 346 (MH+), C₂₁H₁₅NO₄=345 g/mol; HPLC purity=90%.

5.1.2.52 Synthesis of3-(5-(3-hydroxyphenyl)-4-{[4-(2-piperidylethoxy)phenyl]methyl}isoxazol-3-yl)phenol

This compound is synthesized as described in Section 5.1.2.20. Thestarting materials used were 3′-methoxyacetophenone, m-anisoyl chloride,and 4-allyloxybenzyl bromide.

ESMS m/s 471 (MH+), C₂₉H₃₀N₂O₄=470 g/mol; HPLC purity=88.3%.

5.1.2.53 Synthesis of3-(3-hydroxyphenyl)-5-(4-hydroxyphenyl)isoxazol-4-yl4-(2-piperidylethoxy)phenyl ketone

This compound was synthesized as described in Section 5.1.2.20 exceptm-anisoyl chloride as one of the starting materials. The product wasobtained as hydrochloric acid salt in a form of an off-white powder.

¹H NMR (d₆-DMSO) δ 7.84 (d, J=8.76 Hz, 2H, ArH, regioisomer 1), 7.82 (d,J=8.76 Hz, 2H, ArH, regioisomer 2), 7.44 (d, J=8.76 Hz, 2H, ArH,regioisomer 2), 7.36 (d, J=8.76 Hz, 2H, ArH, regioisomer 1), 7.30-7.09(m, 2H, ArH), 7.04-7.02 (m, 2H, ArH), 6.94-6.79 (m, 4H, ArH), 4.41 (brt, 2H, OCH2), 3.46 (br m, 4H, NCH2), 2.79 (br m, 2H, NCH2), 1.80-1.32(m, 6H, CH2); ESMS m/e 485 (MH⁺) , C₂₉H₂₈N₂O₅=484 g/mol; HPLCpurity=100%.

5.1.2.54 Synthesis of2-[3-(4-hydroxyphenyl)-4-phenylisoxazol-5-yl]phenol

This compound was synthesized as described in Section 5.1.2.3, excepto-anisoyl chloride was used as one of the starting materials.

MS m/z 330 (MH+), C₂₁H₁₅NO₃=329 g/mol; HPLC purity=90%. 5.1.2.55Synthesis of 3-[3-(4-hydroxyphenyl)-4-phenylisoxazol-5-yl]phenol

This compound was synthesized as described in Section 5.1.2.54, exceptm-anisoyl chloride was used as one of the starting materials.

MS m/z 330 (MH+), C₂₁H₁₅NO₃=329 g/mol; HPLC purity=90%.

5.1.2.56 Synthesis of 2-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol

This compound was synthesized as described in Section 5.1.2.54, excepto-anisoyl chloride was used as one of the starting materials.

ESMS m/z 282 (MH+), C₁₇H₁₅NO₃=281 g/mol; HPLC purity=90%.

5.1.2.57 Synthesis of 3-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol

This compound was synthesized as described in Section 5.1.2.54, exceptm-anisoyl chloride was used as one of the starting materials.

ESMS m/z 282 (MH+), C₁₇H₁₅NO₃=281 g/mol; HPLC purity=90%.

5.1.2.58 Synthesis of2-[3-(4-hydroxyphenyl)-4-methylisoxazol-5-yl]phenol

This compound was synthesized as described in Section 5.1.2.13, exceptusing o-anisoyl chloride as one of the starting materials.

ESMS m/z 268 (MH+), C₁₆H₁₃NO₃=267 g/mol; HPLC purity=90%.

5.1.2.59 Synthesis of3-[3-(4-hydroxyphenyl)-4-methylisoxazol-5-yl]phenol

This compound was synthesized as described in Section 5.1.2.13, exceptm-anisoyl chloride was used as one of the starting materials.

ESMS m/z 268 (MH+), C₁₆H₁₃NO₃=267 g/mol; HPLC purity=90%.

5.1.2.60 Synthesis of 2-[3-(4-hydroxyphenyl)isoxazol-5-yl]phenol

This compound was synthesized as described in Section 5.1.2.8, excepto-anisoyl chloride was used as one of the starting materials.

ESMS m/z 254 (MH+), C₁₅H₁₁NO₃=253 g/mol; HPLC purity=90%.

5.1.2.61 Synthesis of4-(5-(4-hydroxyphenyl)-4-{[4-(2-piperidylethoxy)phenyl]methyl}isoxazol-3-yl)phenol

This compound was synthesized as described in Section 5.1.2.20. Thestarting materials used are 4′-methoxyacetophenone, p-anisoyl chloride,and 4-allyloxybenzyl bromide.

¹H NMR (d₆-DMSO & CDCl₃): δ 1.8-2.05 (2H, m), 2.62-2.75 (4H, m),2.78-2.85 (2H, m), 3.32-3.38 (2H, m), 3.51 (2H, d, J=11.9 Hz), 3.88 (2H,s), 4.35 (2H, t, J=4.5 Hz), 6.71 (2H, d, J=8.6 Hz), 6.74 (2H, d, J=8.6Hz), 6.76 (2H, d, J=8.8 Hz), 6.96 (2H, d, J=8.6 Hz), 7.22 (2H, d, J=8.6Hz), 7.35 (2H, d, J=8.8 Hz), ESMS m/z 471 (MH+), C₂₉H₃₀N₂O₄=470 g/mol;HPLC purity=85.4%.

5.1.2.62 Synthesis of2-[5-(4-hydroxyphenyl)-4-methylisoxazol-3-yl]phenol

This compound is the regioisomer of the compound synthesized asdescribed in Section 5.1.2.58. Both regioisomers were obtained using themethods described in that Section and the two regioisomers wereseparated by HPLC using a C₁₈ column (Reliasil-BDXC18, 10×50 mm, RaninDynamax) running a first buffer of H₂O/0.1% TFA and a second buffer ofHCN/0.1% TFA through a gradient from 5-95% of the second buffer over anine-minute period at a flow rate of ten ml/min.

ESMS m/z 268 (MH+), C₁₆H₁₃NO₃=267 g/mol; HPLC purity=99%.

5.1.2.63 Synthesis of1-(4-hydroxyphenyl)-4,5-dihydronaphtho[1,2-d]isoxazol-8-ol

This compound was synthesized by following the procedures described inScheme 1 using 7-methoxy-2-tetralone and phenyl 4′-allyloxybenzoate asstarting materials. Demethylation was performed following Method 1 ofStep C.

ESMS m/e 280 (MH⁺), C₁₇H₁₃NO₃=279 g/mol; HPLC purity=92%.

5.1.2.64 Synthesis of3-(4-hydroxyphenyl)-4,5-dihydronaphtho[1,2-c]isoxazol-8-ol

This compound was synthesized as described in Section 5.1.2.29, except7-methoxy-1-tetralone and p-anisoyl chloride were used as startingmaterials.

ESMS m/e 280 (MH⁺), C₁₇H₁₃NO₃=279 g/mol; HPLC purity=90%.

5.1.2.65 Synthesis of3-(4-hydroxyphenyl)-4,5-dihydronaphtho[1,2-c]isoxazol-6-ol

This compound was synthesized as described in Section 5.1.2.29, exceptusing 5-methoxy-1-tetralone and p-anisoyl chloride as startingmaterials.

ESMS m/e 280 (MH⁺), C₁₇H₁₃NO₅=279 g/mol; HPLC purity=90%.

5.1.2.66 Synthesis of 4-[5-(4-hydroxyphenyl)-4-Iodoisoxazol-3-yl]phenol

This compound was synthesized by following Scheme 4.

Steps 1-3 were performed as described for the corresponding stepsdescribed in Section 5.1.2.20 to afford1-[4-bromo-5-(4-methoxyphenyl)isoxazol-3-yl]-4-methoxybenzene.

Step 4: To the above 4-bromoisoxazole in THF at −78° C. was addeddropwise 1.1 eq. of n-BuLi solution (1.6 M in hexane). Maintained thereaction at −78° C. for 1 h followed by addition of 1.1 eq. of I₂ in THFsolution. The reaction was warmed to rt and stirred overnight intosaturated NH₄Cl aq. and extracted with DCM. The organic extracts werewashed with Na₂S₂O₃ aq., brine, dried with MgSO₄ and concentrated invacuo to give1-[4-iodo-5-(3-methoxyphenyl)isoxazol-3-yl]-3-methoxybenzene.

Step 5: Demethylation was following Method 1 described for Step C inScheme 1.

¹H NMR [(CD₃)₂CO]: δ 7.08 (m, 4H), 7.72 (d, J=8.4 Hz, 2H), 8.01 (d,J=8.6 Hz, 2H), 8.86 (s, 1H), 9.10 (s, 1H); ESMS m/z 380 (MH+),C₁₅H₁₀INO₃=379 g/mol; HPLC purity=85.8%.

5.1.2.67 Synthesis of4-[4-chloro-5-(4-hydroxyphenyl]isoxazol-3-yl]phenol

This compound was synthesized as described in Section 5.1.2.66 except,in step 4, N-chlorosuccinimide (NCS) was used as the electrophile totrap the isoxazole anion.

¹H NMR [(CD₃)₂CO]: δ 7.09 (m, 4H), 7.82 (d, J=8.4 Hz, 2H), 7.98 (d,J=9.0 Hz, 2H), 9.70 (br s, 2H); ESMS m/z 288 (MH+), C₁₅H₁₀ClNO₃=287g/mol; HPLC purity=96.0%.

5.1.2.68 Synthesis of 3-(4-hydroxyphenyl)naphtho[1,2-c]isoxazol-8-ol

This compound was synthesized as described in Section 5.1.2.32, except7-methoxy-1-tetralone and p-anisoyl chloride were used as startingmaterials.

ESMS m/e 278 (MH⁺), C₁₇H₁₁NO₃=277 g/mol; HPLC purity=80%.

5.1.2.69 Synthesis of[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N-benzylcarboxamide

This compound was synthesized by following the procedure described inScheme 4.

Steps 1-3 were performed as the corresponding Steps in Section 5.1.2.20to afford 1-[4-bromo-5-(4-methoxyphenyl)isoxazol-3-yl]-4-methoxybenzene.

Step 4: To the above 4-bromoisoxazole in THF at −78° C. was addeddropwise 1.1 eq. of n-BuLi solution (1.6 M in hexane). Maintained thereaction at −78° C. for 1 h following by blowing carbon dioxide gas froma gauge controlled cylinder for ca. 20 min. The reaction was warmed tort carefully, poured into saturated NH₄Cl aq. and extracted with DCM.The organic extracts were washed with brine, dried with MgSO₄ andconcentrated in vacuo to give3,5-bis(3-methoxyphenyl)isoxazole-4-carboxylic acid.

Step 5: Amide bond formation. 4-carboxyisoxazole (obtained from step 4)was dissolved in THF and activated with EDC HCl salt/HOBt/DIEA(1.5:1.5:1.5) and allowed to stand at rt for 5 min. Benzylamine (1.5equiv.) was then added. The reaction was allowed to stand at rtovernight, after which it was diluted with EtOAc and washed with 10%critic acid, 10% NaHCO₃, brine and solvent was removed. Residue waslyophilized in 90% MeCN/H₂O and sampled.

Products were purified by flash chromatography (EtOAc/petroleum ether)to give pure [3,5-bis(4-methoxyphenyl)isoxazol-4-yl]-N-benzamide.

Step 6: Demethylation procedure was described in Method 3 for Step C inScheme 1.

¹N NMR (d₆-DMSO): δ 4.39 (2H, d, J=5.86 Hz), 6.79 (2H, d, J=8.79 Hz),6.83 (2H, d, J=8.79 Hz), 7.22-7.24 (1H, m), 7.26-7.32 (2H, m), 7.50 (2H,d, J=8.61 Hz), 7.56 (2H, d, J=8.79 Hz), 9.20 (1H, t, J=5.95 Hz), 9.87(1H, s), 10.10 (1H, s); ESMS m/z 387 (MH+), C₂₃H₁₈N₂O₄=386 g/mol; HPLCpurity=96.4%.

5.1.2.70 Synthesis of[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N,N-dibutylcarboxamide

This compound was synthesized as described in Section 5.1.2.69 except,in step 5, N,N-di-n-butyl amine was used for amide bond formation.

¹H NMR (d₆-DMSO): δ 0.48 (3H, t, J=7.33 Hz), 0.85 (2H, q, J=7.33 Hz),0.90 (3H, t, J=7.33 Hz), 1.00 (2H, t, J=7.42 Hz), 1.23-1.31 (3H, m),1.51 (2H, t, J=7.24 Hz), 2.86-2.91 (2H, m), 3.43-3.50 (1H, m), 6.86 (2H,d, J=8.61 Hz), 6.90 (2H, d, J=8.79 Hz), 7.51 (2H, d, J=8.79 Hz), 7.58(2H, d, J=8.61 Hz), 9.93 (1 H, s), 10.13 (1H, s); ESMS m/z 409 (M+H).C₂₄H₂₈N₂O₄=408 g/mol; HPLC purity=96.7%.

5.1.2.71 Synthesis of[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N-[3-(2-oxopyrrolidinyl)propyl]carboxamide

This compound was synthesized as described in Section 5.1.2.69 except,in step 5, 1-(3-aminopropyl)pyrrolidin-2-one was used for amide bondformation.

¹H NMR (d₆-DMSO): δ 1.60 (2H, t, J=7.05 Hz), 1.90 (2H, d, J=7.60 Hz),2.19 (2H, d, J=8.06 Hz), 3.10 (2H, t, J=7.14 Hz), 3.16 (2H, q, J=6.59Hz), 3.25 (2H, t, J=6.96 Hz), 3.87 (2H, d, J=8.79 Hz), 7.62 (2H, d,J=8.79 Hz), 7.54 (2H, d, J=8.79 Hz), 8.64 (1H, t, J=5.58 Hz), 9.88 (1H,s), 10.11 (1H, s); ESMS m/z 422 (M+H), C₂₃H₂₃N₃O₅=421 g/mol; HPLCpurity=92.8%.

5.1.2.72 Synthesis of[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N-(2-phenylethyl)carboxamide

This compound was synthesized as described in Section 5.1.2.69 except,in step 5, benzethyl amine was used for amide bond formation.

¹H NMR (d₆-DMSO): δ 2.75 (2H, d, J=7.23 Hz), 7.46 (2H, d, J=6.72 Hz),6.84 (2H, d, J=8.61 Hz), 6.88 (2H, d, J=8.61 Hz), 7.12-7.20 (3H, m),7.23-7.26 (2H, m), 7.49 (2H, d, J=8.61 Hz), 7.56 (2H, d, J=8.79 Hz),8.76 (1H, t, J=5.49 Hz), 9.86 (1H, s), 10.10 (1H, s), ESMS m/z 401(M+H), C₂₄H₂₀N₂O₄=400 g/mol; HPLC purity=82.0%.

5.1.2.73 Synthesis of[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N-[(4-hydroxyphenyl)methyl]carboxamide

This compound was synthesized as described in Section 5.1.2.69 except,in step 5, p-methoxybenzyl amine was used for amide bond formation.

¹H NMR (d₆-DMSO): δ 4.28 (2h, d, J=5.86 Hz), 6.68 (2H, d, J=8.43 Hz),6.79 (2H, d, J=8.79 Hz), 6.82 (2H, d, J=8.79 Hz), 7.03 (2H, d, J=8.24Hz), 7.49 (2H, d, J=8.97 Hz), 7.55 (2H, d,J=8.79 Hz), 9.08 (1H, t,J=5.86 Hz), 9.87 (1H, s), 10.10 (1H, s); ESMS m/z 403 (M+H),C₂₃H₁₈N₂O₅=402 g/mol; HPLC purity=93.0%.

5.1.2.74 Synthesis of[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N-(3-pyridylmethyl)carboxamide

This compound was synthesized as described in Section 5.1.2.69 except,in step 5, 3-pyridylmethylamine was used for amide bond formation.

¹H NMR (d₆-DMSO): δ 4.42 (2H, d, J=5.86 Hz), 6.79 (2H, d, J=8.79 Hz),6.84 (2H, d, J=8.61 Hz), 7.34 (2H, dd, J=7.69 Hz, 4.76 Hz), 7.46 (2H, d,J=8.61 Hz), 7.53 (2H, d, J=8.79 Hz), 7.62 (2H, d, J=7.69 Hz), 9.28 (1H,t, J=5.86 Hz), 9.95 (1H, s), 10.20 (1H, s); ESMS m/z 388 (M+H),C₂₂H₁₇N₃O₄=387 g/mol; HPLC purity=92.7%.

5.1.2.75 Synthesis of[3,5-bis(4-hydroxyphenyl)isoxazol-4yl]-N-(2-pyridylmethyl)carboxamide

This compound was synthesized as described in Section 5.1.2.69 except,in step 5, 2-pyridylmethylamine was used for amide bond formation.

¹H NMR (d₆-DMSO): δ 4.49 (2H, d, J=6.04 Hz), 6.82 (2H, d, J=8.61 Hz),6.86 (2H, d, J=8.61 Hz), 7.21 (1H, d, J=7.88 Hz), 7.29 (1H, dd, J=6.8Hz, 5.0 Hz), 7.55 (2H, d, J=8.79 Hz), 7.63 (2H, d, J=8.79 Hz), 7.71 (1H,td, J=7.5 Hz, 1.65 Hz), 8.51 (1H, d, J=4.03 Hz), 9.29 (1H, t, J=5.86Hz), 9.86 (1H, s), 10.10 (1H, s); ESMS m/z 388 (M+H), C₂₂H₁₇N₃O₄=387g/mol; HPLC purity=91.9%.

5.1.2.76 Synthesis of[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N,N-dimethylcarboxamide

This compound was synthesized as described in Section 5.1.2.69 except,in step 5, N,N-dimethylamine was used for amide bond formation.

¹H NMR (d₆-DMSO): δ 2.72 (3H, s), 6.90 (2H, d, J=8.79 Hz), 6.94 (2H, d,J=8.79 Hz), 7.46 (2H, d, J=8.61 Hz), 7.54 (2H, d, J=8.79 Hz), 10.04 (1H,s), 10.28 (1H, s); ESMS m/z 325 (M+H), C₁₈H₁₆N₂O₄=324 g/mol; HPLCpurity=95.6%.

5.1.2.77 Synthesis of[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N-ethylcarboxamide

This compound was synthesized as described in Section 5.1.2.69 except,in step 5, ethylamine was used for amide bond formation.

¹H NMR (d₆-DMSO): δ 2.49 (3H, t, J=1.83 Hz), 3.18-3.25 (2H, m), 6.86(2H, d, J=8.61 Hz), 6.91 (2H, d, J=8.79 Hz), 7.55 (2H, d, J=8.79 Hz),7.62 (2H, d, J=8.79 Hz), 8.64 (1H, t, J=5.49 Hz), 9.87 (1H, s), 10.10(1H, s); ESMS m/z 325 (M+H), C₁₈H₁₆N₂O₄=324 g/mol; HPLC purity=95.1%.

5.1.2.78 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-(2-pyrrolidinylethoxy)phenyl ketone

This compound was synthesized as described in Section 5.1.2.20 except,in step 6, (2-chloroethyl)pyrroldine was used for alkylation.

LC/MS m/z 471 (MH+), C₂₈H₂₆N₂O₅=470 g/mol; purity=90%.

5.1.2.79 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-(2-morpholin-4-ylethoxy)phenyl ketone

This compound was synthesized as described in Section 5.1.2.20 except,in step 6, 4-(2-chloroethyl)morpholine was used for alkylation.

LC/MS m/z 487 (MH+), C₂₈H₂₆N₂O₆=486 g/mol; purity=90%.

5.1.2.80 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl[3-dimethylamino)propoxy]phenyl ketone

This compound was synthesized as described in Section 5.1.2.20 except,in step 6, (3-chloropropyl)dimethylamine was used for alkylation.

LC/MS m/z 459 (MH+), C₂₇H₂₆N₂O₅=458 g/mol; purity=90%.

5.1.2.81 Synthesis of 4-[3-(diethylamino)propoxy]phenyl3,5-bis(4-hydroxyphenyl)isoxazol-4-yl ketone

This compound was synthesized as described in Section 5.1.2.20 except,in step 6, (3-chloropropyl)diethylamine was used for alkylation.

LC/MS m/z 487 (MH+), C₂₉H₃₀N₂O₅=486 g/mol; purity=90%.

5.1.2.82 Synthesis of[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N-cyclopropylcarboxamide

This compound was synthesized as described in Section 5.1.2.69except, instep 5, cyclopropylamine was used for amide bond formation.

¹H NMR (d₆-DMSO): δ 0.04-0.08 (2H, m), 0.34-0.37 (2H, m), 2.48-2.51 (1Hm), 6.57 (2H, d, J=8.61 Hz), 6.62 (2H, d, J=8.79 Hz), 7.23 (2H, d,J=8.61 Hz), 7.30 (2H, d, J=8.79 Hz), 8.40-8.41 (1H, m), 9.64 (1H, s),9.87 (1H, s); ESMS m/z 337 (M+H, C₁₉H₁₆N₂O₄=336 g/mol; HPLCpurity=81.1%.

5.1.2.83 Synthesis of[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N-cyclobutylcarboxamide

This compound was synthesized as described in Section 5.1.2.69 except,in step 5, cyclobutylamide was used for amide bond formation.

¹H NMR (d₆-DMSO): δ 1.59-1.67 (2H, m), 1.78-1.88 (2H, m), 2.15-2.22 (2H,m), 4.31-4.37 (1H, m), 6.85 (2H, d, J=8.59 Hz), 6.90 (2H, d, J=8.77 Hz),7.54 (2H, d, J=8.59 Hz), 7.60 (2H, d, J=8.77 Hz), 8.88 (1H, d, J=7.59Hz), 9.87 (1H, s), 10.11 (1H, s); ESMS m/z 351 (M+H), C₂₀H₁₈N₂O₄=350g/mol; HPLC purity=98.0%.

5.1.2.84 Synthesis of 4-[2-(diethylamino)ethoxy]phenyl3,5-bis(4-hydroxyphenyl)isoxazol-4-yl ketone

This compound was synthesized as described in Section 5.1.2.20 except,in step 6, (2-chloroethyl)diethylamine was use for alkylation.

LC/MS m/z 473 (MH+), C₂₈H₂₈N₂O₅=472 g/mol; purity=90%.

5.1.2.85 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-[2-dimethylamino)ethoxy]phenyl ketone

This compound was synthesized as described in Section 5.1.2.20 except,in step 6, (2-chloroethyl)dimethylamine was used for alkylation.

LC/MS m/z 445 (MH+), C₂₆H₂₄N₂O₅=444 g/mol; purity=90%.

5.1.2.86 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-{2-[methylbenzylamino]ethoxy}phenyl ketone

This compound was synthesized as described in Section 5.1.2.20 except,in step 6, (2-chloroethyl)methylbenzylamine was used for alkylation.

LC/MS m/z 521 (MH+), C₃₂H₂₈N₂O₅=520 g/mol; purity=90%.

5.1.2.87 Synthesis of2-(4-{[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]carbonyl}phenoxy)-N,N-dimethylacetamide

This compound was synthesized as described in Section 5.1.2.20 except,in step 6, 2-chloro-N,N-dimethylacetamide was used for alkylation.

LC/MS m/z 459 (MH+), C₂₆H₂₂N₂O₆=458 g/mol; purity=90%.

5.1.2.88 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-[2-(1-methylpyrrolidin-2-yl)ethoxy]phenyl ketone

This compound was synthesized as described in Section 5.1.2.20 except,in step 6, 2-(2-chloroethyl)-1-methylpyrrolidine was used foralkylation.

LC/MS m/z 485 (MH+), C₂₉H₂₈N₂O₅=484 g/mol; purity=90%.

5.1.2.89 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-[(1-methyl(3-piperidyl))methoxy]phenyl ketone

This compound was synthesized as described in Section 5.1.2.20 except,in step 6, 2-(2-chloromethyl)-1-methylpiperidine was used foralkylation.

LC/MS m/z 485 (MH+), C₂₉H₂₈N₂O₅=484 g/mol; purity=90%.

5.1.2.90 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-[3-(4-methylpiperazinyl)propoxy]phenyl ketone

This compound was synthesized as described in Section 5.1.2.20 except,in step 6, 1-(2-chloroethyl)-4-methylpiperazine was used for alkylation.

LC/MS m/z 514 (MH+), C₃₀H₃₁N₃O₅=513 g/mol; purity=90%.

5.1.2.91 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-(1-methyl(4-piperidyloxy))phenyl ketone

This compound was synthesized as described in Section 5.1.2.20 except,in step 6, 4-chloro-1-methylpiperidine was used for alkylation.

LC/MS m/z 471 (MH+), C₂₈H₂₆N₂O₅=470 g/mol; purity=90%.

5.1.2.92 Synthesis of3-ethyl-4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol

Synthesis of this compound was performed using the methods described inScheme 7.

Step 1: Synthesis of 1,3-diketone was following the same procedure asfor Step 1 in Section 5.1.2.7 using p-anisyol chloride and1-(2-bromo-4-methoxyphenyl)butan-1-one as starting materials.

Step 2: Isoxazole formation was following the same procedure as for Step4 in Section 5.1.2.7 to afford1-[5-(2-bromo-4-methoxyphenyl)-4-ethylisoxazol-3-yl]-4-methoxybenzene.

Step 3: To a solution of phenylbromoisoxazole (1 eq., obtained fromabove step) in THF at −78° C. was added nBuLi (1.1 eq., 1.6 M inhexane). After the solution was stirred at −78° C. for 1.5 h, it wasadded dropwise into a solution of the bromoethane (1.2 eq.) in THF at−78° C. After 15 min., the solution was allowed to warm to roomtemperature and stirred overnight. After quenching reaction with 1 MHCl, the layers were separated and the aqueous layer extracted withEtOAc (x3). The combined organic layers were washed with saturatedNaHCO₃ (x1) and brine, dried over Na₂SO₄, filtered and concentrated toafford a crude product mixture. Flash chromatography yielded thealkylated product.

Step 4: Demethylation was using Method 1 described for Step C of Scheme1.

¹H NMR (d₆-acetone): δ 7.61 (dd, J=1.8, 8.8 Hz, 2H), 7.21 (d, J=8.3 Hz,1H), 7.00 (dd, J=1.8, 8.8 Hz, 2H), 6.90 (d, J=2.3 Hz, 1H), 6.83 (dd,J=2.3, 8.3 Hz, 1H), 2.55 (q, J=7.4, 2H), 2.54 (q, J=7.4 Hz, 2H), 1.13(t, J=7.4 Hz, 3H), 0.96 (t, J=7.4 Hz, 3H); LC/MS m/z 310 (MH+),C₁₉H₁₉NO₃=309 g/mol; purity=99%.

5.1.2.93 Synthesis of4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-3-methylphenol

The compound was synthesized as described in Section 5.1.2.92. In Step3, iodomethane was used as the electrophile.

¹H NMR (d₆-acetone): δ 7.60 (d, J=8.8 Hz, 2H), 7.22 (d, J=8.8 Hz, 1H),7.00 (d, J=8.8 Hz, 2H), 6.87 (d, J=2.3 Hz, 1H), 6.82 (dd, J=8.3,2.3 Hz,1H), 2.55 (q, J=7.4 Hz, 2H), 2.22 (s, 3H), 0.95 (t, J=7.4 Hz, 3H); LC/MSm/z 296 (MH+), C₁₈H₁₇NO₃=295 g/mol; purity=99%.

5.1.2.94 Synthesis of3-bromo-4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol

This compound was synthesized by following the procedures described forScheme 1 using p-anisyol chloride and1-(2-bromo-4-methoxyphenyl)butan-1-one as starting materials.Demethylation was using Method 1 described for Step C in Scheme 1.

¹H NMR (d₆-acetone): δ 7.60 (d, J=8.8 Hz, 2H), 7.37 (d, J=8.8 Hz, 1H),7.28 (d, J=2.3 Hz), 7.02-6.99 (m, 3H), 2.56 (q, J=7.4 Hz, 2H), 0.96 (t,J=7.4 Hz, 3H); LC/MS m/z 360 (MH+), C₁₇H₁₄BrNO₃=359 g/mol; purity=99%.

5.1.2.95 Synthesis of3-butyl-4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol

The compound was synthesized as described in Section 5.1.2.91. In step3, 1-bromobutane was used as the electrophile.

¹H NMR (d₆-acetone): δ 7.60 (d, J=8.8 Hz, 2H), 7.21 (d, J=8.3 Hz, 1H),7.00 (d, J=8.8 Hz, 2H), 6.89 (d, J=2.3 Hz, 1H), 6.83 (dd, J=8.3, 2.3 Hz,1H), 2.56 (2q, J=7.4 Hz, 4H), 1.53-1.45 (m, 2H), 1.30-1.22 (m, 2H), 0.96(t, J=7.4 Hz, 3H), 0.84 (t, J=7.4 Hz, 3H); LC/MS m/z 338 (MH+),C₂₁H₂₃NO₃=337 g/mol; purity=99%.

5.1.2.96 Synthesis of4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-3-hexylphenol

The compound was synthesized as described in Section 5.1.2.92. In Step3, 1-bromohexane was used as the electrophile.

¹H NMR (d₆-acetone): δ 7.60 (d, J=8.8 Hz, 2H), 7.21 (d, J=8.3 Hz, 1H),6.99 (d, J=8.8 Hz, 2H), 6.90 (d, J=2.3 Hz, 1H), 6.82 (dd, J=8.3,2.3 Hz,1H), 2.56 (2q, J=7.4 Hz, 4H), 1.52-1.46 (m, 2H), 1.29-1.22 (m, 6H), 0.96(t, J=7.4 Hz, 3H), 0.83 (t, J=6.4 Hz, 3H); LC/MS m/z 366 (MH+),C₂₃H₂₇NO₃=365 g/mol; purity=99%.

5.1.2.97 Synthesis of3-(2-bromopropyl)-4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol

The compound was synthesized as described in Section 5.1.2.92. In Step3, 3-bromo-1-propene was used as the electrophile.

¹H NMR (d₆-acetone): δ 7.61 (d, J=8.8 Hz, 2H), 7.28 (d, J=8.3, 1H),7.02-6.99 (m, 3H), 6.92 (dd, J=8.3, 2.7 Hz, 1H), 4.31-4.27 (m, 1H), 3.14(d, J=6.9, 2H), 2.58 (q, J=7.8 Hz, 2H), 1.62 (d, J=6.5 Hz, 3H), 0.98 (t,J=7.8 Hz, 3H); LC/MS m/z 402 (MH+), C₂₀H₂₀BrNO₃=401 g/mol; purity=96%.

5.1.2.98 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazole-4-carbaldehyde

This compound was synthesized based upon Scheme 7.

Step 1-4: Same as corresponding steps in Section 5.1.2.20.

Step 5: Weinreb amide formation. 4-carboxy isoxazole (obtained fromSteps 1-4 above) was dissolved in THF and activated with EDC:HOBt:DIEA(1.5:1.5:1.5) and allowed to stand at rt for 5 min. N-methoxymethylamine(1.5 equiv.) was then added. The reaction mixture was allowed to standat rt overnight, after which it was diluted with EtOAc and washed with10% citric acid, 10% NaHCO₃, and brine. The solvent was removed, and theresidue was purified by flash chromatography (EtOAc/petrol) andlyophilized in 90% MeCN/H₂O to give the desired product as a whitepowder.

Step 6: Reduction of the amide to aldehyde. The Weinreb amide (obtainedfrom step 5) was dissolved in THF and added to a suspension of LiAlH₄ (4equiv.) in THF at −78° C. The reaction was allowed to stir at −78° C.for 2.5 h and was quenched by dropwise addition of 1M HCl. The reactionmixture was then diluted with EtOAc and washed with 10% NaHCO₃ andbrine. After drying over Na₂SO₄, the solvent was removed and the residuewas lyophilized in 90% MeCN/H₂O.

Step 7: Demethylation was performed based upon Method 3 described forstep C in Scheme 1. The above aldehyde was dissolved in DCM and cooledin an ice-bath. BBr₃ (10 equiv.) was added and the reaction was allowedto stir at rt for 5 h. The reaction was then cooled in an ice-bath andquenched with water. EtOAc was added and the reaction was washed withwater and brine. After drying over Na₂SO₄, the solvent was removed andthe residue was purified by flash chromatography (EtOAc/petrol) to yieldpure product.

¹H NMR (d₆-acetone): δ 6.85 (2H, d, J=8.42 Hz), 6.93 (2H, d, J=8.61 Hz),7.54 (2H, d, J=8.42 Hz), 7.88 (2H, d, J=8.61 Hz), 9.81 (1H, s); ESMS m/z282 (M+H), C₁₆H₁₁NO₄=281 g/mol; HPLC purity=97.1%.

5.1.2.99 Synthesis of4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-3-iodophenol

The compound was synthesized as described in Section 5.1.2.92. In Step3, iodine was used as the electrophile.

¹H NMR (d₆-acetone): δ 7.61 (d, J=8.8 Hz, 2H), 7.54 (d, J=2.3 Hz, 1H),7.31 (d, J=8.3 Hz, 1H), 7.05 (dd, J=8.3, 2.3 Hz, 1H), 7.00 (d, J=8.8 Hz,2H), 2.54 (q, J=7.4 Hz, 2H), 0.98 (t, J=7.4 Hz, 3H); LC/MS m/z 408(MH+), C₁₇H₁₄INO₃=407 g/mol; purity=99%.

5.1.2.100 Synthesis of3-chloro-4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol

The compound was synthesized as described in Section 5.1.2.92. In Step3, NCS was used as the electrophile.

¹H NMR (d₆-acetone): δ 7.60 (d, J=8.8 Hz, 2H), 7.39 (d, J=8.8 Hz, 1H),7.10 (d, J=2.8 Hz, 1H), 7.05-6.98 (m, 3H), 2.57 (q, J=7.8 Hz, 2H), 0.96(t, J=7.4 Hz, 3H); LC/MS m/z 316 (MH+), C₁₇H₁₄ClNO₃=315 g/mol;purity=99%.

5.1.2.101 Synthesis of4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-2-fluorophenol

This compound was synthesized by following Scheme 1 using p-anisyolchloride and 1-(3-fluoro-4-methoxyphenyl)butan-1-one as startingmaterials. Demethylation was using Method 1 described for Step C Scheme1.

¹H NMR (d₆-acetone): δ; LC/MS m/z 300 (MH+), C₁₇H₁₄FNO₃=299 g/mol;purity=95%.

5.1.2.102 Synthesis of2-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-5-hydroxybenzoic acid

The compound was synthesized as described in Section 5.1.2.92. In Step3, ethyl chloroformate was used as the electrophile.

¹H NMR (d₆-acetone): δ 7.60-7.54 (m, 3H), 7.38 (s, 1H), 7.20 (d, J=8.3Hz, 1H), 6.99 (d, J=8.8 Hz, 2H), 2.56 (q, J=7.8 Hz, 2H), 0.97 (t, J=7.4Hz, 3H); LC/MS m/z 326 (MH+), C₁₈H₁₅NO₅=325 g/mol; purity=99%.

5.1.2.103 Synthesis of ethyl2-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-5-hydroxybenzoate

The compound was synthesized as described in Section 5.1.2.92. In Step3, ethyl chloroformate was used as the electrophile. Demethylation wasfollowing Method 3 described for Step C in Scheme 1.

¹H NMR (d₆-acetone): δ 7.64 (d, J=8.8 Hz, 2H), 7.49 (d, J=2.8 Hz, 1H),7.34 (d, J=8.3 Hz, 1H), 7.19 (dd, J=8.3, 2.3 Hz, 1H), 7.01 (d, J=8.8 Hz,2H), 4.12 (q, J=7.4 Hz, 2H), 2.54 (q, J=7.4 Hz, 2H), 1.08 (t, J=7.4 Hz,3H), 0.98 (t, J=7.4 Hz, 3H); LC/MS m/z 354 (MH+), C₂₀H₁₉NO₅=353 g/mol;purity=99%.

5.1.2.104 Synthesis of4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-3-(methylsulfinyl)phenol

The compound was synthesized as described in Section 5.1.2.92. In Step3, methyl disulfide was used as the electrophile.

¹H NMR (d₆-acetone): δ 7.66 (d, J=2.3 Hz, 1H), 7.60 (d, J=8.8 Hz, 2H),7.50 (d, J=8.3 Hz, 1H), 7.15 (dd, J=8.3, 2.3 Hz, 1H), 7.01 (d, J=8.8 Hz,2H), 2.83 (s, 3H), 2.64 (q, J=7.4 Hz, 2H), 1.00 (t, J=7.4 Hz, 3H); LC/MSm/z 344 (MH+), C₁₈H₁₇NO₄S=343 g/mol; purity=99%.

5.1.2.105 Synthesis of4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-3-sulfanylphenol

The compound was synthesized as described in Section 5.1.2.92. In Step3, methyl disulfide was used as the electrophile. Demethylation wasfollowing Method 3 described for Step C in Scheme 1.

¹H NMR (d₆-acetone): δ 7.68 (d, J=2.3 Hz, 1H), 7.60 (d, J=8.8 Hz, 2H),7.50 (d, J=8.3 Hz, 1H), 7.15 (dd, J=8.3, 2.3 Hz, 1H), 7.01 (d, J=8.8 Hz,2H), 2.65 (q, J=7.4 Hz, 2H), 1.00 (t, J=7.4 Hz, 3H); LC/MS m/z 314(MH+), C₁₇H₁₅NO₃S=313 g/mol; purity=87%.

5.1.2.106 Synthesis of4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-2-methylphenol

The compound was synthesized as described in Section 5.1.2.92. In Step1, p-anisyol chloride and 1-(3-bromo-4-methoxyphenyl)butan-1-one asstarting materials. In Step 3, iodomethane was used as the electrophile.

¹H NMR (d6-acetone): δ 7.63 (d, J=8.8 Hz, 2H), 7.54 (d, J=8 Hz, 1H),7.42 (s, 1H), 7.04-6.96 (m, 3H), 2.74 (q, J=7.4 Hz, 2H), 2.29, 2.28 (2s,3H), 1.11 (t, J=7.4 Hz, 3H); LC/MS m/z 296 (MH+), C₁₈H₁₇NO₃=295 g/mol;purity=98%.

5.1.2.107 Synthesis of2-butyl-4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol

The compound was synthesized as described in Section 5.1.2.106. In Step3, 1-bromobutane was used as the electrophile.

¹H NMR (d₆-acetone): δ 7.64 (d, J=8.8 Hz, 1H), 7.54 (d, J=8.8 Hz, 2H),7.43 (s, 1H), 7.04-6.98 (m, 3H), 2.74 (q, J=7.4 Hz, 2H), 2.72 (t, J=7.4Hz, 2H), 1.69-1.61 (m, 2H), 1.45-1.38 (m, 2H), 1.12 (t, J=7.4 Hz, 3H),0.95 (t, J=7.4 Hz, 3H); LC/MS m/z 338 (MH+), C₂₁H₂₃NO₃=337 g/mol;purity=99%.

5.1.2.108 Synthesis of 2-ethyl-4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol

The compound was synthesized as described in Section 5.1.2.106. In Step3, bromoethane was used as the electrophile.

¹H NMR (d₆-acetone): δ 7.64 (d, J=8.8 Hz, 2H), 7.54 (d, J=8.8 Hz, 1H),7.44 (d, J=2.3 Hz, 1H), 7.01-6.97 (m, 3H), 2.75 (q, J=7.4 Hz, 2H), 2.73(q, J=7.4 Hz, 2H), 1.24 (t, J=7.4 Hz, 3H), 1.13 (t, J=7.4 Hz, 3H); LC/MSm/z 310 (MH+), C₁₉H₁₉NO₃=309 g/mol; purity=99%.

5.1.2.109 Synthesis of2-bromo-4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol

This compound was synthesized by following Scheme 1 using p-anisyolchloride and 1-(3-bromo-4-methoxyphenyl)butan-1-one as startingmaterials. Demethylation was performed using Method 1 described for StepC in Scheme 1.

¹H NMR (d₆-acetone): δ 7.89 (d, J=2.3 Hz, 1H), 7.64 (d, J=9.2 Hz, 1H),7.54 (d, J=8.8 Hz, 2H), 7.20 (dd, J=1.4, 8.3 Hz, 1H), 7.02 (dd, J=0.9,8.8 Hz, 2H), 2.75 (q, J=7.5 Hz, 2H), 1.12 (t, J=7.9, 3H); LC/MS m/z 360(MH+), C₁₇H₁₄BrNO₃=359 g/mol; purity=99%.

5.1.2.110 Synthesis of4-[3-(4-butanoyloxyphenyl)-4-ethylisoxazol-5-yl]phenyl butanoate

This compound was made as a derivative of the compound preparedaccording to Section 5.1.2.4. To a THF solution of4-[4-ethyl-5-(4-hydroxyphenyl)isoxazol-3-yl]phenol was added butyrylchloride (3.0 eq.) and pyridine (3.0 eq.). The mixture was stirred at rtfor 24 h, poured into cold NaHCO₃, extracted with EtOAc. The organicextracts were washed with brine, dried with Na₂SO₄ and concentrated invacuo to give the product as a white powder.

LC/MS m/z 422 (MH+), C₂₅H₂₇NO₅=421 g/mol; purity=98%.

5.1.2.111 Synthesis of4-[3-(4-acetyloxyphenyl)-4-ethylisoxazol-5-yl]phenyl acetate

This compound was made as a derivative of the compound preparedaccording to Section 5.1.2.4. To a THF solution of4-[4-ethyl-5-(4-hydroxyphenyl)isoxazol-3-yl]phenol was added acetylchloride (3.0 eq.) and pyridine (3.0 eq.). The mixture was stirred at rtfor 24 h, poured into cold NaHCO₃, extracted with EtOAc. The organicextracts were washed with brine, dried with Na₂SO₄ and concentrated invacuo to give the product as a white powder.

LC/MS m/z 366 (MH+), C₂₁H₁₉NO₅=365 g/mol; purity=98%.

5.1.2.112 Synthesis of 3-(4-butanoyloxyphenyl)naphtho[1,2-c]isoxazol-7ylbutanoate

This compound was made as a derivative of the compound prepared asdescribed in Section 5.1.2.32. To a THF solution of3-(4-hydroxyphenyl)naphtho[1,2-c]isoxazol-7-ol was added butyrylchloride (3.0 eq.) and pyridine (3.0 eq.). The mixture was stirred at rtfor 24 h, poured into cold NaHCO₃, extracted with EtOAc. The organicextracts were washed with brine, dried with Na₂SO₄ and concentrated invacuo to give the product as a white powder.

LC/MS m/z 418 (MH+), C₂₅H₂₃NO₅=417 g/mol; purity=98%.

5.1.2.113 Synthesis of 3-(4-acetyloxyphenyl)naphtho[1,2-c]isoxazol-7ylacetate

This compound was made as a derivative of the compound prepared asdescribed in Section 5.1.2.32. To a THF solution of3-(4-hydroxyphenyl)naphtho[1,2-c]isoxazol-7-ol was added acetyl chloride(3.0 eq.) and pyridine (3.0 eq.). The mixture was stirred at rt for 24h, poured into cold NaHCO₃, extracted with EtOAc. The organic extractswere washed with brine, dried with Na₂SO₄ and concentrated in vacuo togive the product as a white powder.

LC/MS m/z 362 (MH+), C₂₁H₁₅NO₃=361 g/mol; purity=98%.

5.1.2.114 Synthesis of 3,5-bis(4-methoxyphenyl)isoxazol-4-yl4-(2-piperidylethoxy)phenyl ketone, chloride

This compound was synthesized by following the methods outlined inScheme 5.

Steps 1-6 were performed as described in Section 5.1.2.20.

Step 7: Formation of HCl salt. The pure product obtained from Step 6 wasdissolved in acetone. To this solution was added at least 5 eq. of conc.HCl aq. and an acetonitrile/H2O (1:1) solution. The mixture was thenlyophilized overnight to give the product as an off-white powder.

LC/MS m/z 360 (MH+), C₁₇H₁₄BrNO₃=359 g/mol; purity=99%.

5.1.2.115 Synthesis of4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-2-hexylphenol

The compound was synthesized as described in Section 5.1.2.106. In Step3, 1-bromohexane was used as the electrophile.

¹H NMR (d₆-acetone): δ 7.63 (d, J=8.8 Hz, 2H), 7.53 (d, J=8.8 Hz, 1H),7.43 (d, J=2.3 Hz, 1H), 7.04-7.97 (m, 3H), 2.76-2.68 (m, 4H), 1.74-1.62(m, 2H), 1.36-1.33 (m, 6H), 1.12 (t, J=7.8 Hz, 3H), 0.91 (t, J=7.4 Hz,3H); LC/MS m/z 366 (MH+), C₂₃H₂₇NO₃=365 g/mol; purity=99%.

5.1.2.116 Synthesis of2-chloro-4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol

The compound was synthesized as described in Section 5.1.2.106. In step3, NCS was used as the electrophile.

¹H NMR (d₆-acetone): δ 7.67-7.50 (m, 4H), 7.20 (d, J=8.3 Hz, 1H), 7.03(d, J=8.8 Hz, 2H), 2.76 (q, J=7.4 Hz, 2H), 1.13 (t, J=7.4 Hz, 3H); LC/MSm/z 315 (MH+), C₁₇H₁₄ClNO₃=314 g/mol; purity=95%.

5.1.2.117 Synthesis of5-[4-ethyl-5-(4-hydroxyphenyl)isoxazol-3-yl]pyridin-2-ol

This compound was synthesized by following Scheme 1, using p-anisyolchloride and methyl-6-methoxypyridine-3-carboxylate as startingmaterials. Demethylation was using Method 1 described for Step C inScheme 1.

¹H NMR (d₆-DMSO) δ 10.04 (one isomer: s, 1H), 9.87 (other isomer: s,1H), 7.78-6.47 (m, 7H), 2.64 (q, J=7.50 Hz, 2H), 1.08 (one isomer: t,J=7.50 Hz, 3H), 1.02 (other isomer: t, J=7.50 Hz, 3H); LC/MS m/z 283(MH+), C₁₆H₁₄N₂O₃=282 g/mol; purity=99%.

5.1.2.118 Synthesis of ethyl5-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-2-hydroxybenzoate

The compound was synthesized as described in Section 5.1.2.106. In Step3, chloro ethylformate was used as the electrophile. Demethylation wasfollowing Method 3 described for Step C in Scheme 1.

¹H NMR (d₆-acetone): δ 7.89 (d, J=8.8 Hz, 1H), 7.65 (d, J=8.8 Hz, 2H),7.55 (d, J=8.8 Hz, 1H), 7.17 (d, J=8.8 Hz, 1H), 7.04 (d, J=8.8 Hz, 2H),4.48 (q, J=7.1 Hz, 2H), 2.76 (q, J=7.4 Hz, 2H), 1.44 (t, J=7.6 Hz, 3H),1.16 (t, J=7.6 Hz, 3H); LC/MS m/z 354 (MH+), C₂₀H₁₈NO₅=353 g/mol;purity=91%.

5.1.2.119 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-methylphenyl ketone

This compound was synthesized by following the procedure described inScheme 7.

Steps, 1-3, were performed as described in Section 5.1.2.20.

Step 4: Again, this step was performed as described in Step 4 of Section5.1.2.20 except p-toluoyl chloride was used for acylation.

Step 5: Demethylation was following Method 3 described for Step C inScheme 1.

¹H NMR (d₆-DMSO): δ 2.26 (3H, s), 6.70 (2H, d, J=8.8 Hz), 6.76 (2H, d,J=9.0 Hz), 7.19 (2H, d, J=8.0 Hz), 7.26 (2H, d, J=8.8 Hz), 7.39 (2H, d,J=9.0 Hz), 7.63 (2H, d, J=8.2 Hz), 9.92 (1H, s,br); ESMS m/z 372 (M+H),C₂₃H₁₇NO₄=371 g/mol; HPLC purity=98%.

5.1.2.120 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl2-chlorophenyl ketone

This compound was synthesized as described in Section 5.1.2.119. In Step4, o-chlorobenzoyl chloride was used for acylation.

¹H NMR (d₆-DMSO): δ 6.67 (2H, d, J=8.8 Hz), 6.75 (2H, d, J=8.8 Hz), 7.18(1H, ddd, J=7.8, 7.2, 1.3 Hz), 7.24-7.27 (1H, m), 7.24 (2H, d, J=8.8Hz), 7.31 (1H, ddd, J=7.8, 7.2, 1.8 Hz), 7.46 (1H, dd, J=7.8, 1.3 Hz),7.51 (2H, d, J=9.0 Hz), 9.77 (1H, s, br); ESMS m/z 392 (M+H),C₂₂H₁₄ClNO₄=391 g/mol; HPLC purity=97%.

5.1.2.121 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl3-chlorophenyl ketone

This compound was synthesized as described in Section 5.1.2.119. In Step4, m-chlorobenzoyl chloride was used for acylation.

¹H NMR (d₆-DMSO): δ 6.71 (2H, d, J=8.8 Hz), 6.77 (2H, d, J=8.6 Hz), 7.25(2H, d, J=8.8 Hz), 7.35 (1H, t, J=7.8 Hz), 7.43 (2H, d, J=8.8 Hz), 7.57(1H, ddd, J=8.0, 2.2, 1.0 Hz), 7.61 (1H, dt, J=7.8, 1.2 Hz), 7.70 (1H,t, J=1.8 Hz), 9.81 (1H, s), 10.14 (1H, s); ESMS m/z 392 (M+H),C₂₂H₁₄ClNO₄=391 g/mol; HPLC purity=86%.

5.1.2.122 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-chlorophenyl ketone

This compound was synthesized as described in Section 5.1.2.119. In Step4, p-chlorobenzoyl chloride was used for acylation.

¹H NMR (d₆-DMSO): δ 6.71 (2H, d, J=8.8 Hz), 6.77 (2H, d, J=8.8 Hz), 7.25(2H, d, J=8.8 Hz), 7.41 (2H, d, J=8.8 Hz), 7.42 (2H, d, J=8.8 Hz), 7.72(2H, d, J=8.8 Hz), 9.82 (1H, s), 10.14 (1H, s); ESMS m/z 392 (M+H),C₂₂H₁₄ClNO₄=391 g/mol; HPLC purity=90%.

5.1.2.123 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl2-fluorophenyl ketone

This compound was synthesized as described in Section 5.1.2.119. In Step4, o-fluorobenzoyl chloride was used for acylation.

¹H NMR (d₆-DMSO): δ 6.86 (2H, d, J=8.8 Hz), 6.77 (2H, d, J=9.0 Hz), 7.01(1H, dd, J=11.1, 8.2 Hz), 7.13 (1H, td, J=7.8, 1.0 Hz), 7.25 (2H, d,J=8.8 Hz), 7.42-7.48 (1H, m), 7.51 (2H, d, J=8.8 Hz), 7.59 (1H, td,J=6.8, 1.8 Hz), 9.89 (1H, s,br); ESMS m/z 376 (M+H), C₂₂H₁₄FNO₄=375g/mol; HPLC purity=98%.

5.1.2.124 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl3-nitrophenyl ketone

This compound was synthesized as described in Section 5.1.2.119. In Step4, m-nitrobenzoyl chloride was used for acylation.

¹H NMR (d₆-DMSO): δ 6.68 (2H, d, J=8.4 Hz), 6.75 (2H, d, J=8.8 Hz), 7.26(2H, d, J=8.4 Hz), 7.47 (2H, d, J=8.6 Hz), 7.59 (1H, t, J=8.0 Hz), 8.06(1H, dd, J=7.8, 1.2 Hz), 8.29 (1H, dd, J=8.2, 2.3 Hz), 8.36 (1H, t,J=2.0 Hz), 9.89 (1H, s,br); ESMS m/z 403 (M+H), C₂₂H₁₄N₂O₆=402 g/mol;HPLC purity=95%.

5.1.2.125 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-nitrophenyl ketone

This compound was synthesized as described in Section 5.1.2.119. In Step4, p-nitrobenzoyl chloride was used for acylation.

¹H NMR (d₆-DMSO): δ 6.70 (2H, d, J=8.8 Hz), 6.76 (2H, d, J=8.8 Hz), 7.25(2H, d, J=8.8 Hz), 7.44 (2H, d, J=8.8 Hz), 7.93 (2H, d, J=9.0 Hz), 8.11(2H, d, J=8.8 Hz), 9.90 (1H, s,br); ESMS m/z 403 (M+H), C₂₂H₁₄N₂O₆=402g/mol; HPLC purity=93%.

5.1.2.126 Synthesis of 3,4-dichlorophenyl3,5-bis(4-hydroxyphenyl)isoxazol-4-yl ketone

This compound was synthesized as described in Section 5.1.2.119. In Step4, 3,4-dichloro-benzoyl chloride was used for acylation.

¹H NMR (d₆-DMSO): δ 6.71 (2H, d, J=8.8 Hz), 6.78 (2H, d, J=9.0 Hz), 7.25(2H, d, J=8.8 Hz), 7.44 (2H, d, J=8.8 Hz), 7.58 (1H, d, J=8.4 Hz), 7.62(1H, dd, J=8.4, 2.0 Hz), 7.88 (1H, d, J=2.0 Hz), 9.82 (1H, s), 10.16(1H, s); ESMS m/z 426 (M+H), C₂₂H₁₃Cl₂NO₄=425 g/mol; HPLC purity=96%.

5.1.2.127 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-butylphenyl ketone

This compound was synthesized as described in Section 5.1.2.119. In Step4, p-n-butylbenzoyl chloride was used for acylation.

¹H NMR (d₆-DMSO): δ 0.80 (3H, t, J=7.4 Hz), 1.16-1.24 (2H, m), 1.40-1.50(2H, m), 2.53 (2H, t, J=7.6 Hz), 6.70 (2H, d, J=8.8 Hz), 6.75 (2H, d,J=9.0 Hz), 7.19 (2H, d, J=8.6 Hz), 7.26 (2H, d, J=8.8 Hz), 7.39 (2H, d,J=8.8 Hz), 7.63 (2H, d, J=8.4 Hz); ESMS m/z 414 (M+H), C₂₆H₂₃NO₄=413g/mol; HPLC purity=92%.

5.1.2.128 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-(tert-butyl)phenyl ketone

This compound was synthesized as described in Section 5.1.2.119. In Step4, p-t-butylbenzoyl chloride was used for acylation.

¹H NMR (d₆-DMSO): δ 1.18 (9H, s), 6.71 (2H, d, J=8.8 Hz), 6.76 (2H, d,J=8.8 Hz), 7.28 (2H, d, J=8.8 Hz), 7.39 (2H, d, J=9.0 Hz), 7.40 (2H, d,J=8.8 Hz), 7.66 (2H, d, J=8.6 Hz), 9.81 (1H, s), 10.10 (1H, s); ESMS m/z414 (M+H), C₂₆H₂₃NO₄=413 g/mol; HPLC purity=98%.

5.1.2.129 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl3-hydroxyphenyl ketone

This compound was synthesized as described in Section 5.1.2.119. In Step4, m-anisoyl chloride was used for acylation.

¹H NMR (d₆-DMSO): δ 6.68-6.72 (1H, m), 6.71 (1H, d, J=8.8 Hz), 6.77 (2H,d, J=8.8 Hz), 6.83 (1H, dd, J=8.2, 0.6 Hz), 7.27 (2H, d, J=8.8 Hz),7.34-7.40 (2H, m), 7.43 (2H, d, J=8.8 Hz), 9.80 (1H, s), 10.11 (1H, s),11.00 (1H, s); ESMS m/z 374 (M+H), C₂₂H₁₅NO₅=373 g/mol; HPLC purity=95%.

5.1.2.130 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl2-hydroxyphenyl ketone

This compound was synthesized as described in Section 5.1.2.119. In Step4, o-anisoyl chloride was used for acylation.

¹H NMR (d₆-DMSO): δ 6.71 (2H, d, J=8.8 Hz), 6.77 (2H, d, J=8.8 Hz), 6.93(1H, ddd, J=7.6, 2.6, 1.6 Hz), 7.11-7.19 (3H, m), 7.27 (2H, d, J=8.8Hz), 7.39 (2H, d, J=8.8 Hz), 9.75 (1H, s), 9.83 (1H, s), 10.13 (1H, s);ESMS m/z 374 (M+H), C₂₂H₁₅NO₅=373 g/mol; HPLC purity=96%.

5.1.2.131 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl phenylketone

This compound was synthesized as described in Section 5.1.2.119. In Step4, benzoyl chloride was used for acylation.

¹H NMR (d₆-DMSO): δ 6.69 (2H, d, J=8.8 Hz), 6.75 (2H, d, J=8.8 Hz), 7.26(2H, d, J=8.8 Hz), 7.40 (2H, d, J=8.8 Hz), 7.53 (1H, t, J=7.5 Hz), 7.72(2H, dd, J=8.3, 1.2 Hz), 9.80 (1H, s), 10.11 (1H, s); ESMS m/z 358(M+H), C₂₂H₁₅NO₄=357 g/mol; HPLC purity=93%.

5.1.2.132 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-methoxyphenyl ketone

This compound was synthesized as described in Section 5.1.2.119. In Step4, p-anisoyl chloride was used for acylation.

¹H NMR (d₆-DMSO): δ 3.77 (3H, s), 6.71 (2H, d, J=9.0 Hz), 6.77 (2H, d,J=9.0 Hz), 6.91 (2H, d, J=8.8 Hz), 7.28 (2H, d, J=9.0 Hz), 7.39 (2H, d,J=9.0 Hz), 7.72 (2H, d, J=9.0 Hz), 9.80 (1H, s), 10.12 (1H, s); ESMS m/z388 (M+H), C₂₃H₁₇NO₅=387 g/mol; HPLC purity=97%.

5.1.2.133 Synthesis of4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-3-phenylthiophenol

The compound was synthesized as described in Section 5.1.2.92. In Step3, phenyl disulfide was used as the electrophile.

¹H NMR (d₆-acetone): δ 7.58 (d, J=8.8 Hz, 2H), 7.42-7.38 (m, 5H), 7.32(d, J=8.3 Hz, 1H), 6.99 (d, J=8.8 Hz, 2H), 6.87 (dd, J=2.8, 8.5 Hz, 1H),6.68 (d, J=2.3 Hz, 1H), 2.56 (q, J=7.4 Hz, 2H), 0.97 (t, J=7.4 Hz, 3H);LC/MS m/z 390 (MH+), C₂₃H₁₉NO₃S=389 g/mol; purity=95%.

5.1.2.134 Synthesis of5-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-2-hydroxybenzamide

The compound was synthesized as described in Section 5.1.2.106. In Step3, tosyl nitrile was used as the electrophile.

¹H NMR (d₆-acetone): δ 7.71 (dd, J=2.3, 8.3 Hz, 1H), 7.64 (d, J=8.8 Hz,2H), 7.54 (d, J=2.3 Hz, 1H), 7.07-6.99 (m, 3H), 2.76 (q, J=7.4 Hz, 2H),1.09 (t, J=7.4 Hz, 3H); LC/MS m/z 325 (MH+), C₁₈H₁₆N₂O₄=324 g/mol;purity=99%.

5.1.2.135 Synthesis of4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-2-phenylthiophenol

The compound was synthesized as described in Section 5.1.2.106. In Step3, phenyl disulfide was used as the electrophile.

¹H NMR (d₆-acetone): δ 7.59-7.36 (m, 9H), 7.14 (d, J=8.3 Hz, 1H), 7.01(d, J=8.8 Hz, 2H), 2.63 (q, J=7.4 Hz, 2H), 1.02 (t, J=7.4 Hz, 3H); LC/MSm/z 390 (MH+), C₂₃H₁₉NO₃S=389 g/mol; purity=99%.

5.1.2.136 Synthesis of4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-2-methylthiophenol

The compound was synthesized as described in Section 5.1.2.106. In Step3, methyl disulfide was used as the electrophile.

¹H NMR (d₆-acetone): δ 7.64 (d, J=8.8 Hz, 1), 7.54 (d, J=8.8 Hz, 2H),7.37 (d, J=2.3 Hz, 1H), 7.07-6.99 (m, 3H), 2.76 (q, J=7.4 Hz, 2H), 2.50,2.47 (2s, 3H), 1.14 (t, J=7.4 Hz, 3H); LC/MS m/z 328 (MH+),C₁₈H₁₇NO₃S=327 g/mol; purity=99%.

5.1.2.137 Synthesis of4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-3-phenylphenol

The compound was synthesized as described in Section 5.1.2.92.

Steps 1 and 2 were performed as described in Section 5.1.2.92.

Step 3: Suzuki coupling was used to introduce the phenyl ring. Theisoxazole obtained from Step 2 was dissolved in toluene and degassedwith argon for 10 min. To this solution was added Pd(PPh₃)₄ (4.0 mol %).After an additional 10 mim; Na₂CO₃ (5 eq.) was added followed by asolution of phenyl boronic acid (1.1 rq.) in ethanol. The reaction washeated to reflux and stirred for 16 h. The reaction mixture was thenpoured into a separatory funnel and the aqueous layer extracted twicewith ethyl acetate. The organic layers were combined, washed with abrine solution and dried over Na₂SO₄. Purification by flashchromatography (40% ethyl acetate/hexanes) yielded the product.

Step 4 Demethylation of the product of Step 3 was performed as describedin Method 3 of Step C in Scheme 1.

¹H NMR (d₆-acetone): δ 7.45-7.40 (m, 3H), 7.32-7.25 (m, 5H), 7.03-7.01(m, 2H), 6.93 (d, J=7.8 Hz, 2H), 2.15 (q, J=7.4 Hz, 2H), 0.63 (t, J=7.4Hz, 3H); LC/MS m/z 358 (MH+), C₂₃H₁₉NO₃=357 g/mol; purity=99%.

5.1.2.138 Synthesis of{4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenyl}(methylsulfonyl)amine

This compound was synthesized by following the procedures describedhere.

Step 1: Synthesis of 1,3-diketone. This was performed as described inStep 1 of Section 5.1.2.7 except 4′-methoxybutyrylphenone and methyl4-nitrobenzoyl chloride were used as the starting materials.

Step 2: Isoxazole formation using the above diketone was performed asdescribed as Step 4 of Section 5.1.2.7.

Step 3: Reduction of nitro group to aniline was achieved by Pd-catalyzedhydrogenation. To a solution of product obtained from Step 2 in ethanolwas added catalytic amount 10% Pd on charcoal. This mixture was stirredunder hydrogen (balloon) overnight, filtered through celite and washedwith ethyl acetate. The filtrate was concentrated in vacuo. The residuewas purified by flash column chromatography to afford product4-[4-ethyl-3-(4-methoxyphenyl)isoxazol-5-yl]phenylamine.

Step 4: To a DCM solution of the above aniline was added methanesulfonylchloride (2.0 eq.) and pyridine (3.0 eq.). The mixture was stirred at rtfor 4 h, poured into water and extracted with ethyl acetate. The organiclayers were combined, washed with brine and dried over Na₂SO₄.Purification by flash chromatography yielded the product{4-[4-ethyl-3-(4-methoxyphenyl)isoxazol-5-yl]phenyl}(methylsulfonyl)amine.

Step 5: Demethylation was following Method 2 described for Step C inScheme 1.

¹H NMR (CDCl₃): δ 7.74-7.50 (m, 4H), 7.32-7.15 (m, 2H), 6.95-6.88 (m,2H), 3.06 (m, 3H), 2.68 (m, 2H), 1.14 (m, 3H); LC/MS m/z 359 (MH+),C₁₈H₁₈N₂O₄S=358 g/mol; purity=99%.

5.1.2.139 Synthesis of5-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-2-methoxybenzamide

This compound was synthesized as described in Section 5.1.2.134. Partialdemethylation of the product afforded5-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-2-methoxybenzamide byfollowing the progress of the reaction using thin-layer chromatography.

¹H NMR (d₆-acetone): δ 8.42 (d, J=2.3 Hz, 1H), 7.84 (dd, J=8.8, 2.9 Hz,1H), 7.65 (d, J=8.8 Hz, 2H), 7.36 (d, J=8.8 Hz, 1H), 7.04 (d, J=8.8 Hz,2H), 4.12, 4.11 (2s, 3H), 2.77 (q, J=7.4 Hz, 3H), 1.15 (t, J=7.4 Hz,3H); LC/MS m/z 339 (MH+), C₁₉H₁₈N₂O₄=338 g/mol; purity=99%.

5.1.2.140 Synthesis of5-[4-ethyl-3-(4-methoxyphenyl)isoxazol-5-yl]-2-hydroxybenzamide

This compound is the regioisomer of the compound prepared as describedin Section 5.1.2.139. Both regioisomers were obtained using the methodsdescribed in that Section and two regioisomers were separated by HPLCusing a C₁₈ column (Reliasil-BDXC18, 10×50 mm, Ranin Dynamax) running afirst buffer of H₂O/0.1% TFA and a second buffer of HCN/0.1% TFA througha gradient from 5-95% of the second buffer over a nine-minute period ata flow rate of ten ml/min.

¹H NMR (d₆-acetone): δ 8.06 (s, 1H), 7.65 (d, J=8.8 Hz, 1H), 7.55 (d,J=8.8 Hz, 2H), 7.45 (d, J=8.8 Hz, 1H), 7.01 (d, J=8.8 Hz, 2H), 4.08,4.07 (2s, 3H), 2.78 (q, J=7.4 Hz, 2H), 1.12 (t, J=7.4 Hz, 3H); LC/MS m/z339 (MH+), C₁₉H₁₈N₂O₄=338 g/mol; purity=99%.

5.1.2.141 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl3-(2-piperidylethoxy)phenyl ketone, chloride

This compound was synthesized as described in Section 5.1.2.20. In Step4, 3-allyloxybenzoyl chloride for acylation.

¹H NMR (d₆-DMSO): δ 10.28 (s, 1H), 9.96 (s, 1H), 7.46 (d, J=8.76 Hz,2H), 7.36-7.30 (m, 5H), 7.24-7.20 (m, 1H), 6.84 (d, J=8.76 Hz, 2H), 6.78(d, J=8.76 Hz, 2H), 4.40-4.30 (br, 2H), 3.50-3.40 (m, 4H), 3.00-2.90 (m,2H), 1.85-1.65 (m, 6H); LC/MS m/z 485 (MH+), C₂₉H₂₈N₂O₅=484 g/mol;purity=95%.

5.1.2.142 Synthesis of5-(4-hydroxyphenyl)-3-(4-methoxyphenyl)isoxazol-4-yl3-(2-piperidylethoxy)phenyl ketone, chloride

This compound was synthesized as described in Section 5.1.2.142. Partialdemethylation afforded the mono-demethylated product by monitoringreaction progress by thin-layer chromatography.

¹H NMR (d₆-DMSO): δ 7.58 (one isomer: d, J=8.76 Hz, 2H), 7.48-7.42(other isomer: m, 2H), 7.40-7.28 (m, 5H), 7.28-7.18 (m, 1H), 7.03 (oneisomer: d, J=8.76 Hz, 2H), 6.97 (other isomer: d, J=8.76 Hz, 2H), 6.83(one isomer: d, J=8.76 Hz, 2H), 6.78 (other isomer: d, J=8.76 Hz, 2H),4.40-4.30 (br, 2H), 3.78 (one isomer: s, 2H), 3.75 (other isomer: s,1H), 3.50-3.40 (br, 4H), 3.00-2.90 (m, 2H), 1.85-1.65 (m, 6H); LC/MS m/z499 (MH+), C₃₀H₃₀N₂O₅=498 g/mol; purity=95%.

5.1.2.143 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl3-(2-pyrrolidinylethoxy)phenyl ketone, chloride

This compound was synthesized as described in Section 5.1.2.141. In Step6, N-(2-chloroethyl)pyrrolidine was used for alkylation.

¹H NMR (d₆-DMSO): δ 10.29 (s, 1H), 9.97 (s, 1H), 7.46 (d, J=8.76 Hz,2H), 7.36-7.30 (m, 5H), 7.25-7.20 (m, 1H), 6.84 (d, J=8.76 Hz, 2H), 6.78(d, J=8.76 Hz, 2H), 4.31 (br, 2H), 3.60-3.45 (m 4H), 3.15-3.00 (m, 2H),2.10-1.80 (m, 4H); LC/MS m/z 471 (MH+), C₂₈H₂₆N₂O₅=470 g/mol;purity=99%.

5.1.2.144 Synthesis of 3-[2-(diethylamino)ethoxy]phenyl3,5-bis(4-hydroxyphenyl)isoxazol-4-yl ketone, chloride

This compound was synthesized as described in Section 5.1.2.141. In Step6, N-(2-chloroethyl)diethylamine was used for alkylation.

¹H NMR (d₆-DMSO): δ 10.31 (s, 1H), 9.98 (s, 1H), 7.46 (d, J=8.76 Hz,2H), 7.36-7.30 (m, 5H), 7.24-7.20 (m, 1H), 6.84 (d, J=8.76 Hz, 2H), 6.78(d, J=8.76 Hz, 2H), 4.33 (br t, 2H), 3.4 (m 2H), 3.19-3.14 (m, 4H),1.25-1.20 (t, J=7.14 Hz, 6H); LC/MS m/z 473 (MH+), C₂₈H₂₈N₂O₅=472 g/mol;purity=99%.

5.1.2.145 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl3-[(1-methyl(3-piperidyl))methoxy]phenyl ketone, chloride

This compound was synthesized as described in Section 5.1.2.141. In Step6, 2-(chloromethyl)-1-methyl-piperidine was used for alkylation.

¹H NMR (d₆-DMSO): δ 10.31 (s, 1H), 9.98 (s, 1H), 7.45 (d, J=8.76 Hz,2H), 7.33-7.29 (m, 5H), 7.20-7.12 (m, 1H), 6.84 (d, J=8.76 Hz, 2H), 6.78(d, J=8.76 Hz, 2H), 3.93-3.74 (m, 2H), 3.50-3.40 (br d, 2H), 2.90-2.70(m, 5H), 2.40-2.25 (m, 1H), 1.90-1.7 (m, 4H); LC/MS m/z 485 (MH+),C₂₉H₂₈N₂O₅=484 g/mol; purity=99%.

5.1.2.146 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl3-[2-(dimethylamino)ethoxy]phenyl ketone, chloride

This compound was synthesized as described in Section 5.1.2.141. In Step6, N-(2-chloroethyl)-dimethylamine was used for alkylation.

¹H NMR (d₆-DMSO): δ 10.35 (s, 1H), 10.02 (s, 1H), 7.45 (d, J=8.76 Hz,2H), 7.37-7.29 (m, 5H), 7.26-7.20 (m, 1H), 6.86-6.83 (d, J=8.76 Hz, 2H),6.85 (d, J=8.76 Hz, 2H), 6.79 (d, J=8.76 Hz, 2H), 4.33 (t, J=4.61 Hz,2H), 3.47 (t, J=4.61 Hz, 2H), 2.80 (s, 6H); LC/MS m/z 445 (MH+),C₂₆H₂₄N₂O₅=444 g/mol; purity=99%.

5.1.2.147 Synthesis of3-(4-hydroxyphenyl)-5-(4-methoxyphenyl)isoxazol-4-yl3-[(1-methyl(3-piperidyl))methoxy]phenyl ketone, chloride

This compound was synthesized as described in Section 5.1.2.145. Partialdemethylation afforded the mono-demethylated product by monitoringreaction progress by thin-layer chromatography.

¹H NMR (d₆-DMSO): δ 10.32 (one isomer: s, 1H), 9.98 (other isomer: s,1H), 7.57 (one isomer: d, J=8.76 Hz, 2H), 7.44 (other isomer: d, J=8.76Hz, 2H), 7.34-7.29 (m, 2H), 7.19-7.17 (m, 1H), 7.03 (one isomer: d,J=8.76 Hz, 2H), 6.97 (other isomer: d, J=8.76 Hz, 2H), 6.84 (one isomer:d, J=8.76 Hz, 2H), 6.80-6.76 (other isomer: d, J=8.76 Hz, 2H), 3.94-3.75(m, 5H), 3.48-3.43 (br d, 2H) 2.90-2.70 (m, 5H), 2.40-2.25 (m, 1H), 1.84(m, 4H); LC/MS m/z 499 (MH+), C₃₀H₃₀N₂O₅=498 g/mol; purity=93%.

5.1.2.148 Synthesis of 3-[2-(dimethylamino)ethoxy]phenyl3-(4-hydroxyphenyl)-5-(4-methoxyphenyl)isoxazol-4-yl ketone, chloride

This compound was synthesized as described in Section 5.1.2.146. Partialdemethylation afforded the mono-demethylated product by monitoringreaction progress by thin-layer chromatography.

¹H NMR (d₆-DMSO): δ 7.59-6.75 (m, 12H), 7.44 (other isomer: d, 2H, J8.76), 4.31 (br, 2H), 3.78 (one isomer: s, 3H), 3.75 (other isomer: s,3H), 3.47 (br, 2H), 2.82 (s, 6H); LC/MS m/z 459 (MH+), C₂₇H₂₆N₂O₅=458g/mol; purity=98%.

5.1.2.149 Synthesis of4-[4-ethyl-3-(4-hydroxy-2-methylphenyl)isoxazol-5-yl]-3-methylphenol

This compound was synthesized by following the procedures described inScheme 1 using 2′-methyl-4′-methoxyacetophenone and2-methyl-4-methoxybenzoyl chloride as starting materials.

¹H NMR (d₆-Acetone): δ 7.24 (d, J=8.29 Hz, 1H, ArH), 7.19 (d, J=8.29 Hz,1H, ArH), 6.87-6.79 (m, 4H, ArH), 2.31 (q, J=7.38 Hz, 2H, CH2), 2.24 (s,3H, CH3), 2.22 (s, 3H, CH3), 0.82 (t, J=7.38 Hz, 3H, CH2CH3); LC/MS m/z310 (MH+), C₁₉H₁₉NO₃=309 g/mol; purity=95%.

5.1.2.150 Synthesis of4-[3-(4-hydroxyphenyl)-4-propylisoxazol-5-yl]phenol

This compound was synthesized based upon Scheme 4.

Step 1-3: Same as the corresponding steps in Section 5.1.2.20.

Step 4: Alkylation. To a solution of 4-bromoisoxazole (1 eq., obtainedfrom step 3) in THF at −78° C. was added nBuLi (1.1 eq., 1.6 M inhexane). After the solution was stirred at −78° C. for 1.5 h, it wasadded dropwise into a solution of the bromopropane (1.2 eq.) in THF at−78° C. After 15 min., the solution was allowed to warm to roomtemperature and stirred overnight. After quenching reaction with 1 MHCl, the layers were separated and the aqueous layer extracted withEtOAc (×3). The combined organic layers were washed with saturatedNaHCO₃ (×1) and brine, dried over Na₂SO₄, filtered and concentrated toafford a crude product mixture. Flash chromatography yielded thealkylated product.

Step 5. Demethylation was performed using Method 3 for Step C in Scheme1.

¹H NMR (CDCl₃/DMSO, 6:1): δ 7.29 (d, J=8.60 Hz, 2H), 7.18 (d, J=8.60 Hz,2H), 6.70 (d, J=8.40 Hz, 2H), 6.68 (d, J=8.40 Hz, 2H), 2.34-2.39 (m,2H), 1.21-1.24 (m, 2H), 0.58 (t, J=7.23 Hz, 3H); ESMS m/z 296 (MH+),C₁₈H₁₇NO₃=295 g/mol; HPLC purity=87.3%.

5.1.2.151 Synthesis of4-[3-(4-hydroxyphenyl)-4-prop-2-enylisoxazol-5-yl]phenol

This compound was synthesized in the same manner as Section 5.1.2.150,except, in step 4, allylbromide was used as the alkylating agent.

¹H NMR (CDCl₃/DMSO, 6:1): δ 9.10 (s, 1H), 8.93 (s, 1H), 7.23 (d, J=8.79Hz, 2H), 7.14 (d, J=8.79 Hz, 2H), 6.59 (d, J=8.79 Hz, 2H), 6.57 (d,J=8.79 Hz, 2H), 5.69-5.78 (m, 1H), 4.87 (d, J=10.36 Hz, 1H), 4.71 (d,J=17.19 Hz, 1H), 3.01-3.02 (m, 2H); ESMS m/z 294 (MH+), C₁₈H₁₅NO₃=293g/mol; HPLC purity=80.8%.

5.1.2.152 Synthesis of 4-[2-(diethylamino)ethylthio]phenyl3,5-bis(4-hydroxyphenyl)isoxazol-4-yl ketone

This compound was synthesized by following the procedures described inScheme 6.

Steps 1-3 were performed as described in Section 5.1.2.20.

Step 4: Performed as described in Step 4 in Section 5.1.2.20, except4-fluorobenzoyl chloride was used for acylation.

Step 5: In a THF suspension of sodium hydride (1.1 eq.) was added2-diethylamino-ethane thiol. The mixture was stirred at rt for 30 min.,followed by addition of the compound obtained from Step 4. The reactionmixture was stirred at rt for 2 h, poured into water, extracted withethyl acetate. The organic extracts were combined, washed with brine,dried with Na₂SO₄, and concentrated in vacuo. Purification by flashcolumn chromatography yielded product4-[2-(diethylamino)ethylthio]phenyl3,5-bis(4-methoxyphenyl)isoxazol-4-yl ketone.

Step 6: Demethylation was performed using Method 2 for Step C in Scheme1.

¹H NMR (d₆- Acetone): δ 7.75 (d, J=8.76 Hz, 2H), 7.57 (d, J=8.76 Hz,2H), 7.45 (d, J=8.76 Hz, 2H), 7.29 (d, J=7.84 Hz, 2H), 6.90 (d, J=8.76Hz, 2H), 6.83 (d, J=8.30 Hz, 2H), 3.10 (t, J=7.83 Hz, 2H), 2.52 (t,J=7.07 Hz, 2H), 0.96 (t, J=7.14 Hz, 2H); LC/MS m/z 489 (MH+),C₂₈H₂₈N₂O₄S=488 g/mol; purity=99%.

5.1.2.153 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-[2-(dimethylamino)ethylthio]phenyl ketone

This compound was synthesized as described in Section 5.1.2.152. In Step5, 2-dimethylamino-ethane thiol was used.

¹H NMR (d₆-Acetone): δ 7.74 (d, J=8.76 Hz, 2H), 7.57 (d, J=9.22 Hz, 2H),7.45 (d, J=8.76 Hz, 2H), 7.27 (d, J=8.76 Hz, 2H), 6.89 (d, J=9.22 Hz,2H), 6.82 (d, J=8.76 Hz, 2H), 3.00 (t, J≦7.14 Hz, 2H), 2.54 (t, J=7.14Hz, 2H), 2.14 (s, 6H); LC/MS m/z 461 (MH+), C₂₆H₂₄N₂O₄S=460 g/mol;purity=95%.

5.1.2.154 Synthesis of 4-(2-azaperhydroepinylethoxy)phenyl3,5-bis(4-hydroxyphenyl)isoxazol-4-yl ketone

This compound was synthesized as described Section 5.1.2.152. In step 5,2-azaperhydroepinylethan-1-ol was used.

¹H NMR (d₆-Acetone): δ 7.82 (d, J=8.76 Hz, 2H), 7.57 (d, J=8.76 Hz, 2H),7.47 (d, J=8.76 Hz, 2H), 6.93-6.88 (m, 4H), 6.84 (d, J=8.76 Hz, 2H),4.10 (t, J=5.99 Hz, 2H) 2.89 (t, J≦5.99 Hz, 2H), 2.72 (t, J=5.53 Hz,2H), 1.65-1.52 (m, 6H); LC/MS m/z 499 (MH+), C₃₀H₃₀N₂O₅=498 g/mol;purity=95%.

5.1.2.155 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-(2-piperidylethoxy)-2-(trifluoromethyl)phenyl ketone

This compound was synthesized as described Section 5.1.2.152. In step 4,2-trifluoromethyl-4-methoxybenzoyl chloride was used. In step 5,N-(2-hydroxyethyl)-piperidine was used.

¹H NMR (d₆-DMSO): δ 7.56 (d, J=8.76 Hz, 1H), 7.54 (d, J=8.76 Hz, 2H),7.27 (d, J=8.76 Hz, 2H), 7.27 (d, J=8.30 Hz, 1H), 6.86 (dd, J=8.76 Hzand 2.31 Hz, 1H), 6.81 (d, J=8.76 Hz, 2H), 6.73 (d, J=8.76 Hz, 2H), 4.12(t, J=5.76 Hz, 2H), 3.31 (br s, 2H), 2.59 (br s, 2H), 2.38 (br m, 4H),1.46 (br m, 4H), 1.37 (br m, 2H); LC/MS m/z 553 (MH+), C₃₀H₂₇F₃N₂O₅=552g/mol; purity=99%.

5.1.2.156 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-{2-[(2-ethylthioethyl)amino]ethoxy}phenyl ketone

This compound was synthesized as described Section 5.1.2.152. In step 5,N-(2-hydroxyethyl)-aziridine was used.

¹H NMR (d₆-Acetone): δ 7.83 (d, J=8.76 Hz, 2H), 7.57 (d, J=9.22 Hz, 2H),7.46 (d, J=9.22 Hz, 2H), 6.94 (d, J=8.76 Hz, 2H), 6.89 (d, J=8.76 Hz,2H), 6.83 (d, J=8.76 Hz, 2H), 4.11 (t, J=5.53 Hz, 2H), 2.98 (t, J=5.53Hz, 2H), 2.82 (t, J=6.50 Hz, 2H), 2.63 (t, J=6.50 Hz, 2H), 2.51 (q,J=7.38 Hz, 2H), 1.17 (t, J=7.38 Hz, 2H); LC/MS m/z 505 (MH⁺),C₂₈H₂₈N₂O₅S=504 g/mol; purity=95%.

5.1.2.157 Synthesis of3-(4-hydroxy-2-methylphenyl)-4,5-dihydronaphtho[1,2-c]isoxazol-7-ol

This compound was synthesized as described in Section 5.1.2.29 using6-methoxy-1-tetralone and 2-methyl-4-methoxybenzoyl chloride as startingmaterials.

¹H NMR (d₆-acetone): δ 7.75 (d, J=8.8 Hz, 1H), 7.30 (d, J=8.3 Hz, 1H),6.86-6.80 (m, 4H), 2.91-2.72 (m, 4H), 2.34 (s, 3H); LC/MS m/z 294 (MH+),C₁₈H₁₅NO₃=293 g/mol; purity=90%.

5.1.2.158 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazole-4-carbonitrile

This compound was synthesized based upon the methodology described inScheme 4.

Steps 1-3 were performed as described in Section 5.1.2.20.

Step 4: was also performed as described in Section 5.1.2.20 except tosylnitrile was used as the electrophile.

Step 5: Demethylation was based upon Method 3 for Step C in Scheme 1.

¹H NMR (d₆-acetone): δ 8.04 (d, J=8.8 Hz, 1H), 7.87 (d, J=8.8 Hz, 1H),7.13 (d, J=8.8 Hz, 1H), 7.06 (d, J=8.8 Hz, 1H); LC/MS m/z 279 (MH+),C₁₆H₁₀N₂O₃=278 g/mol; purity=99%.

5.1.2.159 Synthesis of4-[4-ethyl-5-(4-hydroxyphenyl)isoxazol-3-yl]-3-methylphenol

This compound was synthesized regiospecifically by following proceduresdescribed in Scheme 3.

Step 1: Oxime synthesis. To a suspension of2′-methyl-4′-methoxy-acetophenone (1.0 eq.) in ethanol was addedNH₂OH.HCl (1.2 eq.) and pyridine (1.2 eq.). The mixture was heated toreflux and stirred overnight. The solvent was removed under reducedpressure and the residue was dissolved in EtOAc, washed with water andbrine, dried over Na₂SO₄, filtered and concentrated to afford a crudeproduct mixture. Flash chromatography yielded the oxime as tworegioisomers in 85% yield.

Step 2: isoxazole synthesis. To a solution of oxime obtained from step 1(2 eq.) in THF at 0° C. was added n-BuLi (4.2 eq.). After 30 min, methyl4-methoxybenzoate (1.0 eq.) was added as a solution in THF. Afterstirring for 30 min. at 0° C., the mixture was warmed to roomtemperature. HCl (10 mL, 5 N) was added and the biphasic reactionmixture was heated to reflux and stirred overnight. Upon cooling to 0°C., isoxazole precipitated and was collected via filtration. Thefiltrate was washed with water and the aqueous layer extracted withEtOAc (×3). The organic layers were combined, washed with saturatedNaHCO₃ and brine, dried over Na₂SO₄, filtered and concentrated to afforda crude reaction mixture. Flash chromatography (THF/Hexanes) andrecrystallization (THF/EtOH) yielded the isoxazole.

Step 3 Demethylation was performed as described in Method 3 of Step C ofScheme 1.

¹H NMR (d₆-acetone): δ=7.67 (d, J=8.8 Hz, 2H), 7.15 (d, J=8.4 Hz, 1H),7.03 (d, J=8.8 Hz, 2H), 6.85 (d, J=2.5 Hz, 1H), 6.80 (dd, J=8.2, 2.5 Hz,1H), 2.52 (q, J=7.6 Hz, 2H), 2.18 (s, 3H), 0.98 (t, J=7.6 Hz, 3H); LC/MSm/z 296 (MH=⁺), C₁₈H₁₇NO₃=295 g/mol; purity=99%.

5.1.2.160 Synthesis of3-(4-hydroxy-2-methylphenyl)naphtho[1,2-c]isoxazol-7-ol

This compound was synthesized as described in Section 5.1.2.32 using6-methoxy-1-tetralone and 2-methyl-4-methoxybenzoyl chloride as startingmaterials.

¹H NMR (d₆-acetone): δ 8.31 (d, J=8.3 Hz, 1H), 7.64 (d, J=8.8 Hz, 1H),7.52 (d, J=8.8 Hz, 1H), 7.47-7.38 (m, 3H), 6.94-6.88 (m, 2H), 2.42, 2.38(2s, 3H); LC/MS m/z 292 (MH⁺), C₁₈H₁₃NO₃=291 g/mol; purity=85%.

5.1.2.161 Synthesis of3-(4-hydroxyphenyl)-4,5,6-trihydrobenzo[a]isoxazolo[3,4-c][7]annulen-8-ol

This compound was synthesized in the same manner as Section 5.1.2.29,except in Step 1, 7-methoxy-1-benzosuberone was used to form the1,3-diketone.

ESMS: m/z 294 (MH+), C₁₈H₁₅NO₃=293 g/mol; HPLC purity=80.6%.

5.1.2.162 Synthesis of4-[3-(4-hydroxyphenyl)-4-(2-methoxyphenyl)isoxazol-5-yl]phenol

This compound was synthesized based upon Scheme 4.

Step 1-3: Same as corresponding steps in Section 5.1.2.20.

Step 4: Suzuki coupling of 4-bromoisoxazole with arylboronic acid.Reaction was carried out under an atmosphere of nitrogen and solventswere degassed by bubbling nitrogen for 2 h prior to the reaction.Pd(PPh₃)₄ (0.04 eq) in DMF was added to the 4-bromoisoxazole (obtainedfrom step 3) and 2-methoxyphenylboronic acid (or vinyltributyltin) (1.1eq). Sodium carbonate (0.4 mL, 2M) was then added. The reaction wassealed and allowed to stand overnight at 90° C. Ethyl acetate (20 mL)was then added and the reaction was washed with water and brine. Theorganic fractions were filtered and concentrated under reduced pressure.Residues were lyophilised in 90% MeCN/H₂O. Purification of the compoundwas achieved by teturation with 60% MeCN/H₂O and HPLC purification.

Step 5: Demethylation was following Method 3 described for Step C inScheme 1.

¹H NMR (CDCl₃ & DMSO-d₆): δ 3.04 (3H, s), 6.16 (2H, d, J=8.5 Hz), 6.20(2H, d, J=8.7 Hz), 6.44 (1H, t, J=7.5 Hz), 6.47 (1H, d, J=8.4 Hz), 6.58(1H, dd, J=7.1 Hz, 1.6 Hz), 6.67 (2H, d, J=8.5 Hz), 6.78 (2H, d, J=8.6Hz), 6.87 (3H, t, J=8.3 Hz), 8.69 (1H, s), 8.91 (1H, s); ESMS m/z 360(MH+), C₂₂H₁₇NO₄=359 g/mol; HPLC purity=98.2%.

5.1.2.163 Synthesis of4-{4-[3,5-bis(trifluoromethyl)phenyl]-5-(4-hydroxyphenyl)isoxazol-3-yl}phenol

This compound was synthesized in the same manner as product in Section5.1.2.162, except in Step 4, 3,5-di-trifluoromethylphenylboronic acidwas used for coupling.

¹H NMR (CDCl₃): δ 6.71 (1H, d, J=8.7 Hz), 6.73 (1H, d, J=10.8 Hz), 7.04(1H, d, J=8.4 Hz), 7.20 (1H, d, J=8.4 Hz), 7.60 (2H, s), 7.76 (1H, s),9.25 (1H, s), 9.50 (1H, s); ESMS m/z 466 (MH+), C₂₃H₁₃F₆NO₃=465 g/mol;HPLC purity=86.4%.

5.1.2.164 Synthesis of 3-[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]phenol

This compound was synthesized in the same manner as product in Section5.1.2.162, except in Step 4, 3-methoxyphenylboronic acid was used forcoupling.

¹H NMR (CDCl₃): δ 6.30 (4H, d, J=6.4 Hz), 6.33 (1H, s), 6.35 (1H, d,J=7.7 Hz), 6.44 (1H, d, J=8.1 Hz), 6.80 (1H, t, J=7.8 Hz), 6.85 (2H, d,J=7.6 Hz), 6.95 (2H, d, J=7.8 Hz), 8.64 (1H, s), 8.81 (1H, s), 8.98 (1H,s); ESMS m/z 346 (MH+), C₂₁H₁₅NO₄=345 g/mol; HPLC purity=91%.

5.1.2.165 Synthesis of4-{3-(4-hydroxyphenyl)-4-[3-(trifluoromethyl)phenyl]isoxazol-5-yl}phenol

This compound was synthesized in the same manner as product in Section5.1.2.162, except in Step 4, 3-trifluoromethylphenylboronic acid wasused for coupling.

¹H NMR (CDCl₃): δ 6.13 (2H, d, J=8.5 Hz), 6.17 (2H, d, J=8.7 Hz), 6.67(2H, d, J=8.7 Hz), 6.87 (1H, s), 6.88 (1H, d, J=6.9 Hz), 6.97 (1H, t,J=7.9 Hz), 7.06 (1H, d, J=8.5 Hz), 8.79 (1H, s), 9.02 (1H, s); ESMS m/z398 (MH+), C₂₂H₁₄F₃NO₃=397 g/mol; HPLC purity=99.3%.

5.1.2.166 Synthesis of4-{3-(4-hydroxyphenyl)-4-[4-(trifluoromethyl)phenyl]isoxazol-5-yl}phenol

This compound was synthesized in the same manner as product in Section5.1.2.162, except in step 4, 4-trifluoromethylphenylboronic acid wasused for coupling.

¹H NMR (CDCl₃ & DMSO-d₆): δ 6.34 (2H, d, J=8.4 Hz), 6.38 (2H, d, J=8.4Hz), 6.75 (2H, d, J=8.4 Hz), 6.88 (2H, d, J=8.2 Hz), 6.98 (2H, d, J=8.1Hz), 7.22 (2H, d, J=8.4 Hz), 8.87 (1H, s), 9.09 (1H, s); ESMS m/z 398(MH+), C₂₂H₁₄F₃NO₃=397 g/mol; HPLC purity=98.8%.

5.1.2.167 Synthesis of4-[3-(4-hydroxyphenyl)-4-(2-thienyl)isoxazol-5-yl]phenol

This compound was synthesized in the same manner as product in Section5.1.2.162, except in Step 4, 2-thiophenylboronic acid was used forcoupling.

¹H NMR (CDCl₃ & DMSO-d₆): δ 6.45 (2H, d, J=8.8 Hz), 6.48 (2H, d, J=8.8Hz), 6.69 (1H, dt, J=3.8, 1.2 Hz), 6.79 (1H, dd, J=3.8, 1.2 Hz), 7.00(2H, d, J=8.2 Hz), 7.11 (2H, d, J=8.2 Hz), 7.12-7.15 (1H, m), 8.90 (1H,s), 9.46 (1H, s); ESMS m/z 336 (MH+), C₁₉H₁₃NO₃S=335 g/mol; HPLCpurity=87.9%.

5.1.2.168 Synthesis of4-[3-(4-hydroxyphenyl)-4-(3-thienyl)isoxazol-5-yl]phenol

This compound was synthesized in the same manner as product in Section5.1.2.162, except in Step 4, 3-thiophenylboronic acid was used forcoupling.

¹H NMR (CDCl₃ & DMSO-d₆): δ 6.32 (2H, d, J=8.8 Hz), 6.36 (2H, d, J=8.6Hz), 6.54 (1H, d, J=4.8 Hz), 6.76 (1H, s), 6.82 (2H, d, J=8.5 Hz), 6.94(2H, d, J=8.5 Hz), 7.01-7.03 (1H, m), 8.83 (1H, s), 9.03 (1H, s); ESMSm/z 336 (MH+), C₁₉H₁₃NO₃S=335 g/mol; HPLC purity=94.0%.

5.1.2.169 Synthesis of4-[3-(4-hydroxyphenyl)-4-vinylisoxazol-5-yl]phenol

This compound was synthesized in the same manner as product in Section5.1.2.162, except in Step 4, vinylboronic acid was used for coupling.

¹H NMR (CDCl₃ & DMSO-d₆): δ 5.03 (1H, dd, J=11.4, 1.6 Hz), 5.06 (1H, dd,J=17.8, 1.6 Hz), 6.32 (1H, dd, J=17.8, 11.4 Hz), 6.65 (2H, d, J=9.0 Hz),6.67 (2H, d, J=9.2 Hz), 7.19 (2H, d, J=8.8 Hz), 7.19 (2H, d, J=8.8 Hz),8.95 (1H, s), 9.17 (1H, s); ESMS m/z 280 (MH+), C₁₇H₁₃NO₃=279 g/mol;HPLC purity=93.2%.

5.1.2.170 Synthesis of3-(4-hydroxy-2-methylphenyl)-5-(4-hydroxyphenyl)isoxazol-4-yl4-(2-piperidylethoxy)phenyl ketone

This compound is synthesized as described in Section 5.1.2.20 except, inStep 1, 4′-methoxy-2′-methylacetophenone and p-anisoylchloride are usedas starting materials. A mixture of regioisomers is obtained.

ESMS m/e 499 (MH⁺), C₃₀H₃₀N₂O₅=498 g/mol; HPLC purity=90%.

5.1.2.171 Synthesis of3-(2-ethyl-4-hydroxyphenyl)-5-(4-hydroxyphenyl)isoxazol-4-yl4-(2-piperidylethoxy)phenyl ketone

This compound is synthesized as described in Section 5.1.2.20 except, inStep 1, 4′-methoxy-2′-ethylacetophenone and p-anisoylchloride are usedas starting materials. A mixture of regioisomers is obtained.

ESMS m/e 513 ((MH⁺), C₃₁H₃₂N₂O₅=512 g/mol; HPLC purity=90%.

5.1.2.172 Synthesis of 3,5-bis(4-hydroxy-2-methylphenyl)isoxazol-4-yl4-(2-piperidylethioxy)phenyl ketone

This compound is synthesized as described in Section 5.1.2.20 except, inStep 1, 4′-methoxy-2′-methylacetophenone and 4-methoxy-2-methylbenzoylchloride are used as starting materials.

ESMS m/e 513 (MH⁺), C₃₁H₃₂N₂O₅=512 g/mol; HPLC purity=90%.

5.1.2.173 Synthesis of4-(4-{(hydroxyimino)[4-(2-piperidylethoxy)phenyl]methyl}-5-(4-hydroxyphenyl)isoxazol-3-yl)phenol

This compound is synthesized based upon Scheme 7.

Steps 1-7: Same as corresponding steps in Section 5.1.2.20.

Step 8: Oxime synthesis. To a suspension of the ketone obtained fromStep 7 (1.0 eq.) in ethanol is added NH₂OH.HCl (1.2 eq.) and pyridine(1.2 eq.). The mixture is heated to reflux and stirred overnight. Thesolvent is removed under reduced pressure and the residue is dissolvedin EtOAc, washed with water and brine, dried over Na₂SO₄, filtered andconcentrated to afford a crude product mixture. Flash chromatographyyielded the oxime as a mixture of two regioisomers.

ESMS m/e 500 (MH⁺), C₂₉H₂₉N₃O₅=499 g/mol; HPLC purity=90%.

5.1.2.174 Synthesis of4-(4-{(1E)-2-aza-2-methoxy-1-[4-(2-piperidylethoxy)phenyl]vinyl}-5-(4-hydroxyphenyl)isoxazol-3-yl)phenol

This compound is synthesized in the same manner as the above compoundexcept in Step 8, methoxyamine was used to form the oxime.

ESMS m/e 514 (MH⁺), C₃₀H₃₁N₃O₅=513 g/mol; HPLC purity=90%.

5.1.2.175 Synthesis of 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-[2-(tert-butoxy)ethoxy]phenyl ketone

This compound is synthesized in the same manner as Section 5.1.2.152. Instep 5, 2-(tert-butoxy)ethan-1-ol is used as the nucleophile.

ESMS m/e 474 (MH⁺), C₂₈H₂₇NO₆=473 g/mol; HPLC purity=90%.

5.1.2.176 Synthesis of3-(4-hydroxyphenyl)-5-(4-methoxyphenyl)isoxazol-4-yl4-(2-piperidylethoxy)phenyl ketone

This compound is synthesized based upon Scheme 3 and Scheme 6.

Step 1 and 2: Same as corresponding steps in Section 5.1.2.152 except4′-benzyloxyacetophenone is used to form oxime in Step 1.

Step 3-5: Same as corresponding steps in Section 5.1.2.152 except2-piperidylethan-1-ol is used in Step 5 as the nucleophile.

Step 6: Selective removal of benzyl protecting group. To the aboveobtained compound in MeOH solution was added 10% Pd/C (catalyticamount). The mixture is stirred under hydrogen atmosphere (balloon) atrt overnight, and filtered through celite pad. Ethyl acetate is used towash the residue. The filtrate is concentrated and subjected topurification to yield the title compound.

ESMS m/e 499 (MH⁺), C₃₀H₃₀N₂O₅=498 g/mol; HPLC purity=90%.

5.1.2.177 Synthesis of3,5-bis(4-hydroxyphenyl)-4-{[(4-(2-piperidylethoxy)phenyl]sulfonyl}isoxazole

This compound is synthesized based on Scheme 6.

Steps 1-3: Same as corresponding steps in Scheme 5.1.2.20.

Step 4: Same as Step 4 in Scheme 5.1.2.20 except, 4-fluorophenylsulfonylchloride is used as the electrophile to quench the isoxazole anion.

Step 5: Same as Step 5 in the above example.

Step 6: Demethylation was performed using Method 3 described for Step Cin Scheme 1.

ESMS m/e 521 (MH⁺), C₂₈H₂₈N₂O₆S=520 g/mol; HPLC purity=90%.

5.1.2.178 Synthesis of 3-(4-hydroxyphenyl)isoxazolo[4,3-c]quinolin-7-ol

Step 1: Formation of 1,3-diketone. Same as Step 1 in Section 5.1.2.7except, 2-fluoro-4-methoxyacetophenone and p-anisoyl chloride were usedas starting materials.

Step 2: To a THF solution of the above 1,3-diketone was addeddimethylformamidedimethylacetal (4 eq.) The mixture was heated to refluxfor 16 h and concentrated in vacuo to give a dark residue. Purificationwith column chromatography afforded2-[(dimethylamino)methylene]-3-(2-fluoro-4-methoxyphenyl)-1-(4-methoxyphenyl)propane-1,3-dione.

Step 3: The above intermediate is mixed with ammonia under pressure (abomb equipment) and heated to 60° C. overnight. The reaction is cooledto −78° C. and allowed ammonia to evaporate. To the residue is addedwater and EtOAc. Routine extraction followed by concentration then givesthe crude product, which is purified to give quinolone product.

Step 4: Formation of isoxazole heterocycle. Same as step 4 in Section5.1.2.7 except, using compound obtained above as starting material. Thisstep gives 7-methoxy-3-(4-methoxyphenyl)isoxazolo[4,3-c]quinoline asproduct.

Step 5: Demethylation is performed using Method 3 described for Step Cin Scheme 1.

ESMS m/e 279 (MH⁺), C₁₆H₁₀N₂O₃=278 g/mol; HPLC purity=90%.

5.1.2.179 Synthesis of(2E)-3-(4-{[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]carbonyl}phenyl)prop-2-enoicacid

Step 1-3: Same as corresponding steps in Section 5.1.2.20.

Step 4: Suzuki coupling was used to introduce the phenyl ring. Theisoxazole obtained from step 3 is dissolved in toluene and degassed withargon for 10 min. To this solution is added Pd(PPh₃)₄ (4.0 mol %). Afteran additional 10 min, Na₂CO₃ (5 eq.) is added followed by a solution of4-formylphenyl boronic acid (1.1 rq.) in ethanol. The reaction is heatedto reflux and stirred for 16 h. The reaction mixture is then poured intoa separatory funnel and the aqueous layer extracted twice with ethylacetate. The organic layers are combined, washed with a brine solutionand dried over Na₂SO₄. Purification by flash chromatography (40% ethylacetate/hexanes) then give the product.

Step 5: Honor-Emmons reaction to form α,β-unsaturated ester. The aboveproduct is subjected to Honor-Emmons reaction condition using trimethylphosphonoacetate to afford methyl(2E)-3-{4-[3,5-bis(4-methoxyphenyl)isoxazol-4-yl]phenyl}prop-2-enoate.

Step 6: Saponification (NaOH in water/THF) of the above compound thengives(2E)-3-{4-[3,5-bis(4-methoxyphenyl)isoxazol-4-yl]phenyl}prop-2-enoicacid.

Step 7: Demethylation is performed using Method 3 described for Step Cin Scheme 1.

ESMS m/e 400 (MH⁺), C₂₄H₁₇NO₅=399 g/mol; HPLC purity=90%.

5.1.2.180 Synthesis of7-hydroxy-3-(4-hydroxyphenyl)(4,5-dihydroisoxazolo[4,3-c]quinolin-5-yl)4-(2-piperidylethoxy)phenyl ketone

Steps 1-3: Same as Section 5.1.2.178.

Step 4: Reduction. To a methanol solution of the compound obtained fromstep 3 is added 10% Pd/C. The mixture is stirred under hydrogenatmosphere (balloon) at rt overnight, and filtered through celite pad.Ethyl acetate is used to wash the residue. The filtrate is concentratedand subjected to purification to yield product as7-methoxy-3-[(4-methoxyphenyl)carbonyl]-1,2,3-trihydroquinolin-4-one.

Step 5: Formation of isoxazole heterocycle. Same as step 4 in Section5.1.2.7 except, using compound obtained above as starting material. Thisstep gives7-methoxy-3-(4-methoxyphenyl)-4,5-dihydroisoxazolo[4,3-c]quinoline asthe product.

Step 6: Acylation. To a suspension of sodium hydride (1.1 eq) in THF isadded the above compound (1.0 eq.) at 0° C. The mixture is stirred at rtfor 1 h or until the bubbling stops. To this is added4-(2-piperidylethoxy)benzoyl chloride in THF solution. This mixture isstirred at rt overnight, poured into water followed by routineextraction procedure to give a crude product which is purified to yield7-methoxy-3-(4-methoxyphenyl)(4,5-dihydroisoxazolo[4,3-c]quinolin-5-yl)4-(2-piperidylethoxy)phenyl ketone.

Step 7: Demethylation is performed using Method 3 described for Step Cin Scheme 1.

ESMS m/e 512 (MH⁺), C₃₀H₂₉N₃O₅=511 g/mol; HPLC purity=90%.

5.1.2.181 Synthesis of2-[4-ethyl-5-(4-hydroxyphenyl)isoxazol-3-yl]-5-hydroxybenzenecarbonitrile

Same procedure as Section 5.1.2.92 except, in step 3, tosyl nitrile wasused as the electrophile and in step 4, demethylation was using Method 3for Step C in Scheme 1.

ESMS m/z 307 (MH+), C₁₈H₁₄N₂O₃=306 g/mol, HPLC purity=99%.

5.1.2.182 Synthesis of2-[4-ethyl-5-(4-methoxyphenyl)isoxazol-3-yl]-5-hydroxybenzenecarbonitrile

Partial demethylation yielded of the compound synthesized as describedin Section 5.1.2.181 provided the title compound.

ESMS m/z 321 (MH+), C₁₉H₁₆N₂O₃=320 g/mol, HPLC purity=99%.

5.2 Biological Activity of Compounds of the Invention

5.2.1 In vivo Assays

5.2.1.1 Allen-Doisy Test for Estrogenicity

This test is used to evaluate a test compound for estrogenic activity byobservation of cornification of the vaginal epithelium of inovariectornized rats after administration of a test compound (Allen andDoisy 1923; Mühlbock 1940; Terenius 1971).

Mature female Hsd/Cpb rats, having initial weights between about 150-200g, were obtained from a commercial supplier (Harlan-CPB, Horst, TheNetherlands). The rats were housed in aluminum cages in a light- andtemperature-controlled room (14 hours light/10 hours dark at 19° C.-23°C.). Four rats were housed per cage. The rats were provided free accessto standard pelleted food and to tap water. After a period ofacclimatization (a few days) the rats were ovariectomized bilaterallyunder ether anaesthesia. Vaginal smears were taken over a period of 4-5days. Rats showing positive smears were discarded.

The rats of each treatment group were housed in two juxtaposed cages.Each experiment consisted of 2+n groups of eight rats per group. Tworeference groups received the reference compound (estradiol, 1, 3, 5(10)-estratriene-3, 17-β-diol for subcutaneous (“sc”) administration;ethinylestradiol for oral administration); n groups received the testcompound. For subcutaneous administration, 0.1 μg and 0.2 μg totaldose/rat (approx. 0.4-0.8 μg/kg total dose) was used. For oraladministration, 0.008-0.016 mg total dose/rat (approx. 0.032-0.064 μg/kgtotal dose) was used. Vehicles used for sc administration were (inpreferential order): arachis oil, arachis oil with 100 ml/l benzylalcohol; gelatin (5.0 g/l) and mannitol (50 g/l) in water;methylcellulose (2.0 g/l) and NaCl, (9.0 g/l) in water; or any othersuitable vehicle. For oral administration, the vehicles used were (inpreferential order): gelatin (5.0 g/l) and mannitol (50 g/l) in water;methylcellulose (2.0 g/l) and NaCl, (9.0 g/l) in water; mulgofen (50g/l) (sold under the tradename ELF 719, GAF) and NaCl (9.0 g/l) inwater; or any other suitable vehicle.

Three weeks after ovariectomy, the rats were primed with a single scdose of 1 μg estradiol (in 0.1 ml arachis oil) to ensure maintenance ofsensitivity and greater uniformity of response. In the fourth week, 7days after priming, (preferably on a Monday), the reference or testcompound was administered in 3 equal doses, one in the afternoon of thefirst day of treatment, and two (one in the morning and one in theafternoon) of the second day of treatment. Compound doses were chosenbased on extrapolations of in vitro data obtained in theCHO-transactivation (Section) and/or binding assays for estrogenreceptor using known estrogen agonists and antagonists. For scadministration, the reference compound (estradiol) was administered intotal doses of 0.1-0.2 μg/rat. Test compounds were usually administeredin total doses of 0.01-1.0 mg/rat. Each total sc dose was dividedequally over three administrations, each in a dose volume of 0.25 ml.For oral administration, the reference compound (ethinylestradiol) wasadministered in total doses of 0.008-0.016 mg/rat. Test compounds areusually administered in total doses of 0.01-1.0 mg/rat. Each total oraldose was divided equally over 3 administrations, each in a dose volumeof 0.25 ml. For expression of doses per kg, an average body weight of250 g was assumed. Vaginal smears were taken in the afternoon of thethird day, in the morning and afternoon of the fourth day, and in themorning of the fifth day of the treatment week. Additional vaginalsmears were taken on succeeding days (in the morning) until theestrogenic response was complete. The vaginal smears were made onmicroscope slides. The slides were dried and fixed with 96% ethanol, andstained for about twenty minutes with Giemsa solution (Merck, Darmstadt,Germany), that had been diluted 1:10 with tap water, washed thoroughlyunder tap water, then dried. The percentage of cornified and nucleatedepithelial cells was estimated for each smear was evaluated undermicroscope observation (60×). The rats were allowed to rest for one week(week five of the experiment). The experiment was then repeated, withpriming on the sixth week and administration and observation during theseventh week, as described. The rates were then euthanized under deepanesthesia or with CO₂/O₂ gas.

The developmental phase of the vaginal epithelium for each rat wasevaluated using a scale from “a”-“g” determined as follows (Table 1).The vaginal sequence of normal non-ovariectomized rats with a 4-dayestrous cycle is: diestrus→diestrus→proestrus→estrus. The usual phasesobserved in the mornings of the 4-day estrous cycle using the scale inTable 1 are therefore a, a, e, and g, respectively. The phases b, c, dand f are intermediates.

TABLE 1 Percentage of Percentage of Percentage Of Nucleated CornifiedDevelopmental Leucocytes Epithelial Cells Epithelial Cells Phase >67 % —— a. diestrus 5-50 % >50% — b. late diestrus <5 % >50% — e. proestrus <5% — >50% f. estrus <5 %  <5% >90% g. estrus 5-33 % — >50% d. metestrus33-67 % — <50% c. late metestrus

The number of rats with a positive response is a measure for theestrogenic activity of the test compound. The interpretation of theresults was made as shown in Table 2.

TABLE 2 Percentage of Rats Showing a Positive Response Conclusion 0 %inactive 1%-50 % weakly active >50 % active

5.2.1.2 Anti-Allen-Doisy Test for Anti-Estrogenicity

This test is used to evaluate a test compound for anti-estrogenicactivity when administered in the presence of estrogen (Allen and Doisy1923; Jongh and Laqueur 1938; Mühlbock 1940; Emmens, Cox et al. 1959).More specifically, the ability of the test compound to counteract theestrogenic cornification of vaginal epithelium is determined.

Mature female Cpb rats, having initial weights between about 150-200 g,were obtained from a commercial supplier (CPB-TNO, Zeist, TheNetherlands). The rats were housed in housed in aluminium cages in alight- and temperature-controlled room (14 hours light/10 hours dark at21° C.-23° C.). Four rats were housed per cage. The rats were providedfree access to standard pelleted food and to tap water. After a periodof acclimatization (a few days) the rats were ovariectomized bilaterallyunder ether anaesthesia. Vaginal smears were taken over a period of 4-5days. Rats showing positive smears were discarded.

The rats of each treatment group were housed in two juxtaposed cages.Each experiment consisted of 1+n groups of eight rats per group. Onereference group received the reference compound (nafoxidine HCl); ngroups received the test compound. For oral administration, 0.25mg/rat/day (approx. 1.44 mg/kg/day) was used. Vehicles used forsubcutaneous (“sc”) administration were (in preferential order): arachisoil, arachis oil with 10% benzyl alcohol; gelatin (0.5%) and mannitol(5%) in water; and methylcellulose (0.2%) and NaCl (9.0%) in water. Fororal administration, the vehicles used were (in preferential order):gelatin (0.5%) and mannitol (5%) in water; methylcellulose (0.2%) andNaCl (9.0%) in water; and mulgofen (5%) (sold under the tradename ELF719, GAF) and NaCl (0.9%) in water.

Two weeks after ovariectomy, the rats were primed with a single sc doseof 0.2 μg estradiol (in 0.1 ml arachis oil) administered daily for tendays to ensure maintenance of sensitivity and greater uniformity ofresponse. Administration of estradiol was followed immediately byadministration of test compound or vehicle. Test compounds wereadministered at 1.0 mg/rat. For sc administration, the dose volume was0.1 ml; for oral administration, the dose volume was 0.25 ml. Vaginalsmears were taken daily throughout the administration period. Thevaginal smears were made on microscope slides. The slides were dried andfixed with 96% ethanol, and stained for about twenty minutes with Giemsasolution (Merck, Darmstadt, Germany), that had been diluted 1:10 withtap water, washed thoroughly under tap water, then dried. The percentageof cornified and nucleated epithelial cells was estimated for each smearwas evaluated under microscope observation (60×). Following theexperiment, rats were euthanized under deep anesthesia or with CO₂/O₂gas.

The developmental phase of the vaginal epithelium for each rat wasevaluated using a scale from “a”-“g” determined as follows (Table 3).The vaginal sequence of normal non-ovariectomized rats with a 4-dayestrous cycle is: diestrus→diestrus→proestrus→estrus. The usual phasesobserved in the mornings of the 4-day estrous cycle using the scale inTable 3 are therefore a, a, e, and g, respectively. The phases b, c, dand f are intermediates.

TABLE 3 Percentage of Percentage of Percentage Of Nucleated CornifiedDevelopmental Leucocytes Epithelial Cells Epithelial Cells Phase >67 % —— a. diestrus 5-50 % >50% — b. late diestrus <5 % >50% — e. proestrus <5% — >50% f. estrus <5 %  5% >90% g. estrus 5-33 % — >50% d. metestrus33-67 % — <50% c. late metestrus

Smears showing any of phases e, f, or g were considered to be estrogenic(i.e., the vaginal epithelium showed cornification). The final resultwas expressed as a ratio of the number of smears showing estrogenicresponse to the total number of smears collected from the third daythrough the final day of the study. The number of rats with a positiveresponse is a measure for the anti-estrogenic activity of the testcompound. The interpretation of the results was made as shown in Table4.

TABLE 4 Percentage of Rats Showing a Positive Response Conclusion >70 %inactive 35%-70 % weakly active <35 % active

5.2.1.3 Immature Rat Uterotrophic Bioassay for Estrogenicity andAnti-Estrogenicity

Antiestrogenic activity is determined by the ability of a test compound(3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 4-(2-piperidylethoxy)phenylketone) to suppress the increase in uterine wet weight resulting fromthe administration of 0.2 μg 17-β-estradiol (“E₂”) per day. Anystatistically significant decreases in uterine weight in a particulardose group as compared with the E₂ control group are indicative ofanti-estrogenicity.

One hundred sixty (160) female pups (10 per foster dam) were received at12 days of age. One hundred forty (140) female pups (19 days old) in the35-50 g body weight range were selected for the study. On day 19 of age,when the pups weighed approximately 35-50 g, they were body weight-orderrandomized into treatment groups. Observations for mortality, morbidity,availability of food and water, general appearance and signs of toxicitywere made twice daily. Pups not used in the study were euthanized alongwith the foster dams. Initial body weights were taken just prior to thestart of treatment at day 19 of age. The final body weights were takenat necropsy on day 22 of age.

Treatment commenced on day 19 of age and continued until day 20 and 21of age. Each animal was given three subcutaneous (“sc”) injections dailyfor 3 consecutive days. Three rats in each of the control and mid- tohigh-level dose test groups were anesthetized with a ketamine/xylazinemixture. Their blood was collected by exsanguination using a 22 gaugeneedle and 5 ml syringe flushed with 10 USP units sodium heparin/mlthrough the descending vena cava; and then transferred into a 5 ml greentop plasma tube (sodium heparin (freeze-dried), 72 USP units). Plasmasamples were collected by centrifugation, frozen at −70° C., andanalyzed using mass spectrographic to determine the presence and amountof test compound in the serum. Blood chemistry was also analyzed todetermine other blood parameters. The uteri from the rats were excisedand weighed. The remaining rats were sacrificed by asphyxiation underCO₂. The uteri from these rats were excised, nicked, blotted to removefluid, and weighed to the nearest 0.1 mg.

In order to determine whether the test compound significantly affectedfinal body weight, a parametric one-way analysis of variance (ANOVA) wasperformed (SIGMASTAT version 2.0, available commercially from JandelScientific, San Rafael, Calif.). Estrogen agonist and antagonistactivity was assessed by comparing uterine wet weights across treatmentgroups using a parametric ANOVA on logic transformed data. The data weretransformed to meet assumptions of normality and homogeneity of varianceof the parametric ANOVA. The F value was determined to be significant(p<0.05) and a Student-Newman-Kuels multiple range test was performed todetermine the presence of significant differences among the treatmentgroups. Since the final body weights were significantly different acrosstreatment groups (p=0.999), uterine weight:body weight ratios were notcompared.

The test compound was determined to act as a mixed extrogenagonist/antagonist. The uterotrophic response was submaximal compared tothat seen with the reference estrogen, 17β-estradiol. Submaximaluterotrophic response, even at elevated dose levels, is characteristicof partial estrogen agonists. When the test compound was tested incombination with 17β-estradiol, partial inhibition of the uterotrophicresponse was observed at all three dose levels; hence, the test compoundpossessed estrogen antagonist activity. However, the test compound didnot completely inhibit the 17β-estradiol-stimulated uterotrophicresponse; implying that it is a mixed estrogen agonist/antagonist inthis bioassay.

5.2.1.4 Estrogen Receptor Antagonist Efficacy in MCF-7 Xanograft Model

MCF-7 human mammary tumors from existing in vivo passages are implantedsubcutaneously into 95 female Ncr-nu mice. A 17-β-estradiol pellet(Innovative Research of America) is implanted on the side opposite thetumor. Both implants are performed on the same day.

Treatment is started when the tumor sizes are between 75 and 200 mg.Tumor weight is calculated according to the formula for the volume of anellipsoid, $\frac{l \times w^{2}}{2}$

where l and w are the larger- and smaller dimensions of the tumor andunit tumor density is assumed. The test compounds are administered BID:q7h×2, with one drug preparation per week. The test compounds are storedat +4° C. between injections. The dose of test compound is determinedaccording to the individual animal's body weight on each day oftreatment. Gross body weights are determined twice weekly, starting thefirst day of treatment. Mortality checks are performed daily. Micehaving tumors larger than 4,000 mg, mice having ulcerated tumors, as andmoribund mice are sacrificed prior to the day of study termination. Thestudy duration is limited to 60 days from the day of tumor implantationbut termination could occur earlier as determined to be necessary.Terminal bleeding of all surviving mice is performed on the last day ofthe experiment. Statistical analysis is performed on the data gathered,including mortality, gross individual and group average body weights ateach weighing, individual tumor weights and median group tumor weight ateach measurement, the incidence of partial and complete regressions andtumor-free survivors, and the calculated delay in the growth of themedian tumor for each group.

5.2.1.5 OVX Rat Model

This model evaluates the ability of a compound to reverse the decreasein bone density and increase in cholesterol levels resulting fromovariectomy (Black, Author et al. 1994; Willson, Author et al. 1997).Three-month old female rats are ovariectomized (“ovx”), and testcompounds are administered daily by subcutaneous route beginning one daypost-surgery. Sham operated animals and ovx animals with vehicle controladministered are used as control groups. After 28 days of treatment, therats are weighed, the overall body weight gains obtained and the animalseuthanized. Blood bone markers (e.g., osteocalcin and bone-specificalkaline phosphatase), total cholesterol, and urine markers (e.g.,deoxypyridinoline and creatinine) are measured. Uterine wet weights arealso obtained. Both tibiae and femurs are removed from the test animalsfor peripheral quantitative computed tomography scanning or othermeasurement of bone mineral density. Data from the ovx and test vehicleanimals are compared to the sham and ovx control animals to determinetissue specific estrogenic/antiestrogenic effects of the test compounds.

5.2.2 In vitro Assays

5.2.2.1 ERα Binding Assays

ERα receptor (˜0.2 mg/ml, Affinity Bioreagents) was diluted to about2×10⁻³ mg/ml in phosphate-buffered saline (“PBS”) at a pH of 7.4. Fiftymicroliters l of the ERα-PBS solution was then added to each the wellsof a flashplate (Wallac SCINTISCTRIPS). The plates were sealed andstored in the dark at 4° C. for 16-18 hours. The buffered receptorsolution is removed just prior to use, and the plates were washed 3times with 200 microliters per well of PBS. The washing was typicallyperformed using a slow dispense of reagent into the wells to avoidstripping the receptor from the well surface.

For library screening, 150 microliters of 1 nM ³H-estradiol (New EnglandNuclear, Boston, Mass.) in 20 mM Tris-HCl, 1 mM EDTA, 10% glycerol, 6 mMmonothioglycerol, 5 mM KCl, pH 7.8 was mixed with 50 microliters of thetest compound (in same buffer) in a 96 well microtiter plate (Costar3794), resulting in a final estradiol concentration of 0.6 nM. Inaddition, several dilutions of estradiol, centered on the IC₅₀ of 1-2 nMwere also added to individual wells to generate a standard curve. Theplates were gently shaken to mix the reagents. A total of 150microliters from each of the wells is added to the corresponding wellsof the pre-coated ERα plates. The plates were sealed (Packard #6005185)and the components in the wells were incubated either at roomtemperature for 4 hours or at 4° C. overnight. The receptor bound ligandwas read directly after incubation using a scintillation counter(TRILUX, Wallac). The amount of receptor bound ligand was determineddirectly, i.e., without separation of bound from free ligand. Ifestimates of both bound and free ligand were required, the supernatantwas removed from the wells, liquid scintillant added, and the wellscounted separately in a liquid scintillation counter.

5.2.2.2 ERβ Binding Assays

ERβ receptor (˜0.2 mg/ml, Affinity Bioreagents) was diluted to about2×10⁻³ mg/ml in phosphate-buffered saline (“PBS”) at a pH of 7.4. Fiftymicroliters of the ERβ-PBS solution was then added to each the wells ofa flashplate (Wallac SCINTISCTRIPS). The plates were sealed and storedin the dark at 4° C. for 16-18 hours. The buffered receptor solution isremoved just prior to use, and the plates were washed 3 times with 200microliters per well of PBS. The washing was typically performed using aslow dispense of reagent into the wells to avoid stripping the receptorfrom the well surface.

For library screening, 150 microliters of 1 nM ³H-estradiol (New EnglandNuclear, Boston, Mass.) in 20 mM Tris-HCl, 1 mM EDTA, 10% glycerol, 6 mMmonothioglycerol, 5 mM KCl, pH 7.8 was mixed with 50 microliters of thetest compound (in same buffer) in a 96 well microtiter plate (Costar3794), resulting in a final estradiol concentration of 0.6 nM. Inaddition, several dilutions of estradiol, centered on the IC₅₀ of 1-2 nMwere also added to individual wells to generate a standard curve. Theplates were gently shaken to mix the reagents. A total of 150microliters from each of the wells is added to the corresponding wellsof the pre-coated ERβ plates. The plates were sealed (Packard #6005185)and the components in the wells were incubated at room temperatureeither for 4 hours or at 4° C. overnight. The receptor bound ligand wasread directly after incubation using a scintillation counter (TRILUX,Wallac). The amount of receptor bound ligand was determined directly,i.e., without separation of bound from free ligand. If estimates of bothbound and free ligand were required, the supernatant was removed fromthe wells, liquid scintillant added, and the wells counted separately ina liquid scintillation counter.

5.2.2.3 ERα/ERβ Transactivation Assays

5.2.2.3.1 Construction of Transfected CHO Cells

The above-mentioned transfected CHO cells were derived from CHO KI cellsobtained from the American Type Culture Collection (“ATCC”, Rockville,Md.). The transfected cells were modified to contain the following fourplasmid vectors: (1) pKCRE with DNA for the human estrogen receptor, (2)pAG-60-neo with DNA for the protein leading to neomycin resistance, (3)pRO-LUC with DNA for the rat oxytocin promoter and for fireflyluciferase protein, and (4) pDR2 with DNA for the protein leading tohygromycine resistance. All transformations with these geneticallymodified CHO cells were performed under rec-VMT containment according tothe guidelines of the COGEM (Commissie Genetische Modificatie).Screening was performed either in the absence of estradiol(estrogenicity) or in the presence of estradiol (anti-estrogenicity).

Reagents

The following reagents were prepared using ultra pure water (milli-Qquality):

1. Culture Medium

Dulbecco's MEM/HAM F12 powder (12.5 g/l; Gibco, Paisley, UK) wasdissolved in water. Sodium bicarbonate (2.5 grams/liter (“g/l”)),L-glutamine (0.36 g/l) and sodium pyruvate (5.5×10⁻² g/l) were added.This medium was supplemented with an aqueous mixture (0.50 ml/l medium)of ethanolamine (2.44 ml/l), sodium selenite (0.9 mg/l), and2-mercaptoethanol (4.2 ml/l). The pH of the medium was adjusted to7.0±0.1 with NaOH or HCl (1 mol/l), and the medium was sterilized bymembrane filtration using a filter having 0.2 μm pores. The resultingserum-free culture medium was stored at 4° C.

2. Antibiotics Solution

Streptomycin sulfate (25 g; Mycofarm, Delft, The Netherlands) and sodiumpenicillin G (25 g; Mycofarm) were dissolved in 1 l water and sterilizedby membrane filtration using a filter having 0.2 μm pores.

3. Defined Bovine Calf Serum Supplemented (“DBCSS”)

DBCSS (Hyclone, Utah), sterilized by the manufacturer, was inactivatedby heating for 30 min at 56° C. with mixing every 5 min. Aliquots of 50ml and 100 ml were stored at −20° C.

4. Charcoal-Treated DBCSS (“cDBCSS”)

Charcoal (0.5 g; Norit A) was washed with 20 ml water (3 times) and thensuspended in 200 ml Tris buffer. For coating 0.05 g dextran (T70;Pharmacia, Sweden) is dissolved in a suspension that was stirredcontinuously for 3 hours at room-temperature. The resultingdextran-coated charcoal suspension was centrifuged for 10 min at 8,000N/kg. The supernatant was removed and 100 ml DBCSS was added to theresidue. The suspension was stirred for 30 min at 45° C. under asepticconditions. Following stirring, the charcoal was removed bycentrifugation for 10 min at 8000 N/kg. The supernatant was sterilizedby membrane filtration using a first filter having a pore size of 0.8 μmfollowed by filtration with a second filter having a pore size of 0.2μm. The sterilized, heat-inactivated cDBCSS was stored at −20° C.

5. Tris Buffer

Tromethamine (“Tris”, 1.21 g; 10 mmol) was dissolved in approximately950 ml water. The solution pH was adjusted to 7.4 using HCl (0.2 mol/l)and the volume raised to 1 l with additional water. This buffer wasprepared fresh prior to use.

6. Luclite Substrate Solution

Luclite luminescence kit, developed for firefly luciferase activitymeasurements in microtiter plates was obtained from a commercial source(Packard, Meriden, Conn.). Ten milliliters of the above-described buffersolution was added to each flask of substrate.

Preparation of Transfected Cells

Under aseptic conditions, the above-described culture medium wassupplemented with antibiotics solution (2.5 ml/l) and heat-inactivatedcDBCSS (50 ml/l) to give complete medium. One vial of theabove-described recombinant CHO cells was taken from the seed stock inliquid nitrogen and allowed to thaw in water at approximately 37° C. ARoux flask (80 cc) was inoculated with about 5×10⁵ viable cells/ml incomplete medium. The flask was flushed with 5% CO₂ in air until a pH of7.2-7.4 resulted. The cells were subsequently incubated at 37° C. Duringthis period, the complete medium was refreshed twice.

Following incubation the cell culture was trypsinized and inoculated at1:10 dilution in a new flask (180 cc cell culturing) and at 5×10³ cellswith 100 μl complete medium per well in a 96-well white culture platefor transactivation assays. The 96 well plates were incubated over twodays. The cells were grown as a monolayer at the bottoms of the wellsand reached confluence after two days. After a cell culture period of 20passages, new cells were taken from the seed stock in liquid nitrogen.

5.2.2.3.2 Assay of Compounds

Assay for Estrogenicity

Experiments were performed in groups of three blocks, each block in aseparate microtiter plate. Each block included the following four groups

Group Contents 1 One trasactivation group of four wells, each containingethanol and transfected cells. This group was used to estimate totaltransactivation. 2 One total transactivation group of four wellscontaining beta-estradiol (1 × 10⁻⁷ M) and transfected cells. This groupwas used to estimate total transactivation of cells. 3 Three standardgroups of five wells each, containing five different concentrations ofnon-transfected and transfected cells. 4 Test or reference compoundgroups (n groups, n ≦ 21) of three wells each, contaning three differentconcentrations of test or reference compound and transfected cells.

Aliquots of ten μl of control, standard, test, and reference compoundswere added by pipette into wells of the relevant groups as definedabove. Each of the wells included 190 μl of complete medium.

Group Contents 1 Ethanol 2 Standard solution in ethanol (10⁻⁹ M, to beraised to 10⁻⁶ M final concentration). 3 Standard solutions in ethanol(0.47 × 10⁻¹¹ M, 0.95 × 10⁻¹¹ M, 1.95 × 10⁻¹¹ M, 3.9 × 10⁻¹¹ M, and 7.8× 10⁻¹¹ M, to be raised to 0.47 × 10⁻⁸ M, 0.95 × 10⁻⁸ M, 1.95 × 10⁻⁸ M,3.9 × 10⁻⁸ M, and 7.8 × 10⁻⁸ M respectively). 4 Test or referencecompound in six different concentrations 1 × 10⁻⁵ M, 3.16 × 10⁻⁶ M, 1 ×10⁻⁶ M, 3.16 × 10⁻⁷ M, 1 × 10⁻⁷ M, 3.16 × 10⁻⁸ M, respectively.

Assay for Anti-Estrogenicity

Experiments were performed in groups of three blocks, each block in aseparate microtiter plate. Each block included the following fourgroups, each group containing estradiol, 1,3,5(10)-estratriene-3,17-β-diol (10⁻¹⁰ M) in the final reaction mixture.

Group Contents One transactivatin group of four wells, each containingethanol and transfected cells. This group was used to estimate totaltransactivation. 2 One group of completely inhibited transactivationgroup of four wells containing ICI 164,384 (10⁻⁶ M) and transfectedcells. This group was used to estimate complete inhibition oftransactivation. 3 Three standard groups of five wells each, containingfive different concentrations of non-transfected and transfected cells.4 Test or reference compound groups (n groups, n ≦ 21) of three wellseach, containing three different concentrations of test or referencecompound and transfected cells.

Aliquots of ten μl of control, standard, test, and reference compoundswere added by pipette into wells of the relevant groups as definedabove. Each of the wells included 190 μl of complete medium.

Group Contents 1 Ethanol 2 Standard solution in ethanol (10⁻⁹ M, to beraised to 10⁻⁶ M final concentration). 3 Standard solution in ethanol(0.47 × 10⁻¹¹ M, 0.95 × 10⁻¹¹ M, 1.95 × 10⁻¹¹ M, 3.9 × 10⁻¹¹ M, and 7.8× 10⁻¹¹ M, to be raised to 0.47 × 10⁻⁸ M, 0.95 × 10⁻⁸ M, 1.95 × 10⁻⁸ M,3.9 × 10⁻⁸ M, and 7.8 × 10⁻⁸ M respectively). 4 Test or referencecompound in six different concentrations 1 × 10⁻⁵ M, 3.16 × 10⁻⁶ M, 1 ×10⁻⁶ M, 3.16 × 10⁻⁷ M, 1 × 10⁻⁷ M, 3.16 × 10⁻⁸ M, respectively.

The microtiter plates were shaken for at least 15 minutes to ensuredissolution of all compounds. Simultaneously, 100 μl estradiol,1,3,5(10)-estratriene-3,17-β-diol (10⁻⁷ M) was added to 40 ml ofcomplete medium, shaken, and equilibrated to 37° C. About 100 μl of thissolution was added to microtiter white culture plates seeded theprevious day with 10⁴ transfected cells in 100 μl of complete medium.The microtiter white culture plates were gently shaken for at least 15minutes and incubated for 16 h at 37° C. in the dark under a humidifiedatmosphere flushed with 5% CO₂ in air.

Finally, 200 μl of complete medium was removed from the microtiterculture plates, while 50 μl of LUCLITE substrate solution was added tothe remaining 50 μl of medium and cells. After ten minutes cell, celllysis was substantially complete. After sealing the top of the plate,luciferase activity was measured with a luminescence counter. Eachsample was counted once for 2.5 s using a scintillation (luminescence)counter. All luminescence measurements were recorded on a teleprinter.

5.2.2.3.3 Evaluation of Responses

The counting figures are corrected to a standardized plate and convertedinto numbers of light flashes per second (“cps”). For each block(microtiter plate), the mean cps values for the total and non-specifictransactivation groups were calculated. For each concentration ofstandard (separate for each well), test and reference compound, thepercentage of transactivation activity relative to the maximum specificestradiol, 1,3,5(10)-estratriene-3,17-β-diol transactivation activitywas calculated using the formula: $\frac{\begin{matrix}{{{cps}\left( {{{standard}/{test}}\quad {compound}} \right)} -} \\{{mean}\quad {cps}\text{(non-specific~~transactivation)}}\end{matrix}}{\begin{matrix}{{{cps}\left( {{total}\quad {transactivation}} \right)} -} \\{{mean}\quad {cps}\text{(non-specific~~transactivation)}}\end{matrix}} \times 100.$

The percentage in the three blocks was evaluated statistically using theanalysis of a 3-point parallel line assay in blocks. In order to meetbetter the requirements for this analysis, the percentages were replacedby their logit values. The log concentration-response curves for thestandard, test, and reference compounds were tested for linearity; andthe latter curves also for parallelism with the curve for the standardcompound. If no significant curvature and no significant deviation fromparallelism at the 0.01 levels were found; then the relativetransactivation activity of the test compound with respect to estradiol,1,3,5(10)-estratriene-3,17-β-diol (potency ratio), together with the 95%confidence interval, was calculated. For antagonist assays, the relativeinhibitory potency of transactivation activity of the test compound withrespect to the standard antagonist, ICI 164,384 was calculated. Forcompounds showing significant agonist or antagonist activity in theseinitial screens, more accurate EC₅₀ values were determined by generatingtwelve-point curves with 3-fold dilutions of the compounds. In thiscase, the range of concentrations was selected based on the compoundactivity in the initial screens.

The following compounds of the invention were determined to be active(i.e., have agonist or antagonist values of EC₅₀≧4×10⁻⁶ M (ERα) and/orEC₅₀≧4×10⁻⁶ M (ERβ)) against either or both ERα and ERβ:4-{5-[2-(4-hydroxyphenyl)ethyl]-4-benzylisoxazol-3-yl}phenol,4-[4-ethyl-5-(phenoxymethyl)isoxazol-3-yl]phenol,4-[5-(4-hydroxyphenyl)-4-phenylisoxazol-3-yl]phenol,4-[4-ethyl-5-(4-hydroxyphenyl)isoxazol-3-yl]phenol,4-{5-[2-(4-hydroxyphenyl)ethyl]-4-phenylisoxazol-3-yl}phenol,4-[5-(4-hydroxyphenyl)-4-benzylisoxazol-3-yl]phenol,4-[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]phenol,4-[5-(4-hydroxyphenyl)isoxazol-3-yl]phenol,4-{5-(4-hydroxyphenyl)-4-[4-(2-piperidylethoxy)phenyl]isoxazol-3-yl}phenol,4-{4-(4-hydroxyphenyl)-3-[4-(2-piperidylethoxy)phenyl]isoxazol-5-yl}phenol,3-[4,5-bis(4-hydroxyphenyl)isoxazol-3-yl]phenol,2-[4,5-bis(4-hydroxyphenyl)isoxazol-3-yl]phenol,4-[3-(4-hydroxyphenyl)-4-methylisoxazol-5-yl]phenol,4-[3-(4-hydroxyphenyl)-5-phenylisoxazol-4-yl]phenol,4-[5-(4-fluorophenyl)-3-(4-hydroxyphenyl)isoxazol-4-yl]phenol,4-{4-(4-hydroxyphenyl)-5-[3-(trifluoromethoxy)phenyl]isoxazol-3-yl}phenol,4-{4-(4-hydroxyphenyl)-5-[4-(trifluoromethoxy)phenyl]isoxazol-3-yl}phenol,4-[5-(4-hydroxyphenyl)-4-(phenoxymethyl)isoxazol-3-yl]phenol,4-[5-(4-hydroxyphenyl)-4-(phenylthiomethyl)isoxazol-3-yl]phenol,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-(2-piperidylethoxy)phenyl ketone,5-(4-hydroxyphenyl)-3-(4-methoxyphenyl)isoxazol-4-yl4-(2-piperidylethoxy)phenyl ketone,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-[2-(hydroxypiperidyl)ethoxy]phenyl ketone,3-(4-hydroxyphenyl)-5-(4-methoxyphenyl)isoxazol-4-yl4-[2-(hydroxypiperidyl)ethoxy]phenyl ketone,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 4-hydroxyphenyl ketone,4-hydroxyphenyl 3-(4-hydroxyphenyl)-5-(4-methoxyphenyl)isoxazol-4-ylketone, 4-[4-bromo-5-(4-hydroxyphenyl)isoxazol-3-yl]phenol,4-[4-(bromomethyl)-5-(4-hydroxyphenyl)isoxazol-3-yl]phenol,4-{5-(4-hydroxyphenyl)-4-[(4-hydroxyphenoxy)methyl]isoxazol-3-yl}phenol,4-[4-(hydroxymethyl)-5-(4-hydroxyphenyl)isoxazol-3-yl]phenol,4-(7-methoxy-4,5-dihydronaphtho[1,2-c]isoxazol-3yl)phenol,3-(4-hydroxyphenyl)-4,5-dihydronaphtho[1,2-c]isoxazol-7-ol,3-[3-(4-hydroxyphenyl)isoxazol-5-yl]phenol,3-(4-hydroxyphenyl)naphtho[1,2-c]isoxazol-7-ol,3-(3-hydroxyphenyl)-4,5-dihydronaphtho[1,2-c]isoxazol-7-ol,3-(2-hydroxyphenyl)-4,5-dihydronaphtho[1,2-c]isoxazol-7-ol,3-[5-(3-hydroxyphenyl)isoxazol-3-yl]phenol,2-[3-(3-hydroxyphenyl)isoxazol-5-yl]phenol,2-[5-(2-hydroxyphenyl)isoxazol-3-yl]phenol,3-[3-(3-hydroxyphenyl)-4-methylisoxazol-5-yl]phenol,2-[3-(3-hydroxyphenyl)-4-methylisoxazol-5-yl]phenol,2-[3-(2-hydroxyphenyl)-4-methylisoxazol-5-yl]phenol,3-[4-ethyl-3-(3-hydroxyphenyl)isoxazol-5-yl]phenol,2-[4-ethyl-3-(3-hydroxyphenyl)isoxazol-5-yl]phenol,2-[4-ethyl-3-(2-hydroxyphenyl)isoxazol-5-yl]phenol,3-[4-(4-hydroxyphenyl)-5-(3-hydroxyphenyl)isoxazol-3-yl]phenol,2-[3-(3-hydroxyphenyl)-4-(4-hydroxyphenyl)isoxazol-5-yl]phenol,2-[4-(4-hydroxyphenyl)-5-(2-hydroxyphenyl)isoxazol-3-yl]phenol,3-(2-hydroxyphenyl)-4,5-dihydronaphtho[2,1-d]isoxazol-7-ol,2-(7-methoxy-4,5-dihydronaphtho[1,2-c]isoxazol-3-yl)phenol,2-[5-(3-hydroxyphenyl)-4-methylisoxazol-3-yl]phenol,2-[4-ethyl-5-(3-hydroxyphenyl)isoxazol-3-yl]phenol,2-[4-(4-hydroxyphenyl)-5-(3-hydroxyphenyl)isoxazol-3-yl]phenol,3-(5-(3-hydroxyphenyl)-4-{[4-(2-piperidylethoxy)phenyl]methyl}isoxazol-3-yl)phenol,, 2-[3-(4-hydroxyphenyl)-4-phenylisoxazol-5-yl]phenol,3-[3-(4-hydroxyphenyl)-4-phenylisoxazol-5-yl]phenol,2-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol,3-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol,2-[3-(4-hydroxyphenyl)-4-methylisoxazol-5-yl]phenol,3-[3-(4-hydroxyphenyl)-4-methylisoxazol-5-yl]phenol,2-[3-(4-hydroxyphenyl)isoxazol-5-yl]phenol,4-(5-(4-hydroxyphenyl)-4-{[4-(2-piperidylethoxy)phenyl]methyl}isoxazol-3-yl)phenol,2-[5-(4-hydroxyphenyl)-4-methylisoxazol-3-yl]phenol,1-(4-hydroxyphenyl)-4,5-dihydronaphtho[1,2-d]isoxazol-8-ol,3-(4-hydroxyphenyl)-4,5-dihydronaphtho[1,2-c]isoxazol-8-ol,3-(4-hydroxyphenyl)-4,5-dihydronaphtho[1,2-c]isoxazol-6-ol,4-[5-(4-hydroxyphenyl)-4-iodoisoxazol-3-yl]phenol,4-[4-chloro-5-(4-hydroxyphenyl)isoxazol-3-yl]phenol,3-(4-hydroxyphenyl)naphtho[1,2-c]isoxazol-8-ol,[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N-benzylcarboxamide,[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N,N-dibutylcarboxamide,[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N-[3-(2-oxopyrrolidinyl)propyl]carboxamide,[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N-(2-phenylethyl)carboxamide,[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N-[(4-hydroxyphenyl)methyl]carboxamide,[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N-(3-pyridylmethyl)carboxamide,[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N-(2-pyridylmethyl)carboxamide,[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N,N-dimethylcarboxamide,[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N-ethylcarboxamide,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 4-(2-pyrrolidinylethoxy)phenylketone, 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-(2-morpholin-4-ylethoxy)phenyl ketone,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 4-[3-(dimethylamino)propoxy]phenylketone, 4-[3-(diethylamino)propoxy]phenyl3,5-bis(4-hydroxyphenyl)isoxazol-4-yl ketone,[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N-cyclopropylcarboxamide,[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]-N-cyclobutylcarboxamide,4-[2-(diethylamino)ethoxy]phenyl 3,5-bis(4-hydroxyphenyl)isoxazol-4-ylketone, 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-[2-(dimethylamino)ethoxy]phenyl ketone,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-{2-[methylbenzylamino]ethoxy}phenyl ketone,2-(4-{[3,5-bis(4-hydroxyphenyl)isoxazol-4-yl]carbonyl}phenoxy)-N,N-dimethylacetamide,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-[2-(1-methylpyrrolidin-2-yl)ethoxy]phenyl ketone,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-[(1-methyl(3-piperidyl)methoxy]phenyl ketone,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl4-[3-(4-methylpiperazinyl)propoxy]phenyl ketone,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 4-(1-methyl(4-piperidyloxy))phenylketone, 3-ethyl-4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol,4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-3-methylphenol,3-bromo-4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol,3-butyl-4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol,4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-3-hexylphenol,3-(2-bromopropyl)-4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol,3,5-bis(4-hydroxyphenyl)isoxazole-4-carbaldehyde,4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-3-iodophenol,3-chloro-4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol,4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-2-fluorophenol,2-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-5-hydroxybenzoic acid,ethyl 2-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-5-hydroxybenzoate,4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-3-(methylsulfinyl)phenol,4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-3-sulfanylphenol,4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-2-methylphenol,2-butyl-4-[4-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol,2-ethyl-4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol,2-bromo-4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol,4-[3-(4-butanoyloxyphenyl)-4-ethylisoxazol-5-yl]phenyl butanoate,4-[3-(4-acetyloxyphenyl)-4-ethylisoxazol-5-yl]phenyl acetate,3-(4-butanoyloxyphenyl)naphtho[1,2-c]isoxazol-7-yl butanoate,3-(4-acetyloxyphenyl)naphtho[1,2-c]isoxazol-7-yl acetate,3,5-bis(4-methoxyphenyl)isoxazol-4-yl 4-(2-piperidylethoxy)phenylketone, chloride,4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-2-hexylphenol,2-chloro-4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenol, ethyl5-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-2-hydroxybenzoate,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 4-methylphenyl ketone,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 2-chlorophenyl ketone,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 3-chlorophenyl ketone,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 4-chlorophenyl ketone,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 2-fluorophenyl ketone,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 3-nitrophenyl ketone,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 4-nitrophenyl ketone,3,4-dichlorophenyl 3,5-bis(4-hydroxyphenyl)isoxazol-4-yl ketone,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 4-butylphenyl ketone,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 4-(tert-butyl)phenyl ketone,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 3-hydroxyphenyl ketone,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 2-hydroxyphenyl ketone,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl phenyl ketone,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 4-methoxyphenyl ketone,4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-3-phenylthiophenol,5-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-2-hydroxybenzamide,4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-2-phenylthiophenol,4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-2-methylthiophenol,4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-3-phenylphenol,{4-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]phenyl}(methylsulfonyl)amine,5-[4-ethyl-3-(4-hydroxyphenyl)isoxazol-5-yl]-2-methoxybenzamide,5-[4-ethyl-3-(4-methoxyphenyl)isoxazol-5-yl]-2-hydroxybenzamide,2-[4-ethyl-3-(4-methoxyphenyl)isoxazol-5-yl]-5-hydroxybenzamide,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 3-(2-piperidylethoxy)phenylketone, chloride, 5-(4-hydroxyphenyl)-3-(4-methoxyphenyl)isoxazol-4-yl3-(2-piperidylethoxy)phenyl ketone, chloride,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 3-(2-pyrrolidinylethoxy)phenylketone, chloride, 3-[2-(diethylamino)ethoxy]phenyl3,5-bis(4-hydroxyphenyl)isoxazol-4-yl ketone, chloride,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl3-[(1-methyl(3-piperidyl))methoxy]phenyl ketone, chloride,3,5-bis(4-hydroxyphenyl)isoxazol-4-yl 3-[2-(dimethylamino)ethoxy]phenylketone, chloride, 3-(4-hydroxyphenyl)-5-(4-methoxyphenyl)isoxazol-4-yl3-[(1-methyl(3-piperidyl))methoxy]phenyl ketone, chloride,3-[2-(dimethylamino)ethoxy]phenyl3-(4-hydroxyphenyl)-5-(4-methoxyphenyl)isoxazol-4-yl ketone, chloride,4-[4-ethyl-3-(4-hydroxy-2-methylphenyl)isoxazol-5-yl]-3-methylphenol,4-[3-(4-hydroxyphenyl)-4-propylisoxazol-5-yl]phenol, and4-[3-(4-hydroxyphenyl)-4-prop-2-enylisoxazol-5-yl]phenol.

5.2.2.4 MCF-7 Cell Proliferation Assays

This assay determines the estrogen agonist/antagonist activity of a testcompound by the effect of the test compound on the proliferation ofMCF-7 cells as measured by the incorporation of 5-bromo-2′-deoxyuridine(“BrdU”) in a chemiluminescent assay format.

MCF-7 cells (ATCC HTB-22) were maintained in log-phase culture usingDMEM/HamF12 medium (v/v 1/1) that had been supplemented with 10% fetalbovine serum (“FBS”), at 37° C., and under at 5% CO₂ atmosphere. Thecells were plated in a 96-well plate at a density of 7,000 cells perwell. After 24 hours, the cells were further incubated in phenolred-free DMEM/HamF12 medium supplemented with 10% FBS that had beenfiltered with dextran-coated charcoal to deplete endogenous estrogen(DCC-FBS). The cells were incubated in this medium for an additional 24hours, at which time either test compound at varying concentrations todetermine the IC₅₀ for the compound. Each test compound was incubatedwith the cells either in the absence of estradiol (detection of estrogenagonist activity) or in the presence of 1 nM estradiol (detection ofestrogen antagonist activity).

The cells were cultured in the presence of test compounds for 24 hoursat 37° C. and under a 5% CO₂ atmostphere. Cell proliferation wasdeteteced by measuring the level of BrdU incorporation into DNA. Thiswas accomplished using a commercially available reagent kit (BoeringerMannheim/Roche). The assay was run according to the manufacturersdirection. Ten microliters of BrDU labeling reagent, diluted accordingto the manufacturers directions was added directly into each well, andincubation was continued for four hours. The culture media was thenaspirated from the wells, and 100 μl of the fixing/denaturing agent fromthe kit was added. The cells were fixed for 30 minutes at roomtemperature. The plates were aspirated again, and 100 μl ofperoxidase-labeled anti-BrdU antibody from the kit was added to eachwell. After one hour, the plates were washed six times with phosphatebuffered saline (“PBS”), and 100 μl of SUPERSIGNAL (a chemilumiscentperoxidase substrate, Pierce Chemical) was added. The plates were shakenfor ten minutes at room temperature, and the resulting chemiluminescentsignals were counted using a TRILUX scintillation counter. Two compoundsof the invention,4-{5-(4-hydroxyphenyl)-4-[4-(2-piperidylethoxy)phenyl]isoxazol-3-yl}phenoland 3,4-bis(4-hydroxyphenyl)isoxazol-4-yl-4-(2-piperidylethoxy)phenylketone, were tested using the above-described protocol, were determinedto have IC₅₀ values of less than 600 nM in the presence of 1 nMestradiol.

Thus, the present invention will be seen to provide new compounds thathave strong estrogen receptor-modulating action. These compounds can beemployed in compositions and methods for treating estrogenreceptor-mediated disorders, such as osteoporosis, breast andendometrial cancer, Alzheimer's disease, and atherosclerosis.

The disclosure above is for the purposes of illustration and notlimitation. Those having skill in the arts relevant to the presentinvention (e.g., the organic chemistry, medicianl chemistry,endocrinology, and medical arts) will appreciate from the foregoing thepresent invention encompasses many additional embodiments of theinvention that are not described explicitly, but which nevertheless areprovided by the teachings of the present invention. Such additionalembodiments include, but are not limited to, estrogen reeptor-mediateddiseases other than osteporosis, breast and endometrical cancer,Alzheimers's disease, and atherosclerosis, that are preventable ortreatable using the compounds, compositions, and methods of theinvention. Still other aspects include compounds that can be designed,synthesized, and tested for therapeutic or prophylactic effect using theteachings of the foregoing disclosure.

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What is claimed is:
 1. A compound having the formula:

and its pharmaceutically acceptable salts, wherein: X₃ and X₄ areselected independently from the group consisting of nitrogen and oxygensuch that if one of X₃ and X₄ is nitrogen, then the other of X₃ and X₄is oxygen to form thereby an isoxazole ring structure; X₅ is—(X₁₀)_(n)—, wherein n is an integer between 1 and 3 and X₁₀, for eachvalue of n, is selected indenpendently from the group consisting ofoxygen, —SO_(x)— where x is and integer between 0 and 2, nitrogen,nitrogen substituted with optionally substitued loweralkyl, aryl,aralkyl, heteroaryl, heteroaralkyl, arylcarbonyl, alkylcarbonyl,aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, andmethylene or methine, each optionally substituted from the groupconsiting of halo, cyano, nitro, thio, amino, carboxyl, formyl, andoptionally substituted loweralkyl, loweralkylcarbonyloxy,arylcarbonyloxy, heteroarylcarbonyloxy, cycloalkylcarbonyloxy,cycloheteroalkylcarbonyloxy, aralkycarbonyloxy,heteroaralkylcarbonyloxy, (cycloalkyl)alkylcarbonyloxy,(cycloheteroalkyl)alkylcarbonyloxy, loweralkylcarbonyl, arylcarbonyl,heteroarylcarbonyl, cycloalkycarbonyl, cycloheteroalkycarbonyl,aralkycarbonyl, heteroaralkylcarbonyl, (cycloalkyl)alkylcarbonyl,(cycloheteroalkyl)alkylcarbonyl, loweralkylaminocarbonyl,arylaminocarbonyl, aralkylaminocarbonyl, heteroarylaminocarbonyl,heteroaralkylaminocarbonyl, loweralkylcarbonylamino, arylcarbonylamino,heteroarylcarbonylamino, cycloalkylcarbonylamino,cycloheteroalkylcarbonylamino, aralkylcarbonylamino,heteroaralkylcarbonylamino, (cycloalkyl)alkylcarbonylamino,(cycloheteroalkyl)alkylcarbonylamino, loweralkylamino, arylamino,aralkylamino, heteroarylamino, heteroaralkylamino, loweralkylsulfonyl,arylsulonyl, heteroarylsuflony, cycloalkylsulfonyl,aralkycarbonylthioooxy, carbonylythio, heteroaralkylcarbonylthio,(cycloalkyloxycarbonylthio, (cycloheteroalkyl)alkylcarbonylthio,loweralkyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,cycloalkyloxycarbonyl, cycloheteroalkyloxycarbonyl,aralyoxycarbonyloxloxycarbonyl, heteroaralkyloxycarbony,(cycloalkyl)alkyloxycarbony, (cycloheteroalkyl)alkyloxycarbony,iminoloweralkyl, iminocycloalky, ininocycloheteroalkyl, iminoaralkyl,iminoheteroaralkyl, (cycloalkyl)iminoalky, and(cycloheteroalkyl)iminoalkyl; X₆-X₉ selected independently from thegroup consisting of oxygen, sulfur, sulfinyl, nitrogen, and optionallysubstituted methine; and R₄ is selected from the group consisting ofoptionally substitued loweralkyl, aryl, heteraryl, cycloalky,cycloheteroalkyl, aralkyl, heteraralkyl, (cycloalkyl)alkyl, and(cycloheteroalkyl)alkyl.
 2. The compound of claim 1, wherein n is 1 andX₁₀ is selected from the group consisting of nitrogen, optionallysubstitued nitrogen, and optionally substituted methylene or methine. 3.The compound of claim 2, wherein R₄ is selected from the groupconsisting of optinally substituted aryl, heteroaryl, aralkyl, andheteroaralkyl.
 4. The compound of claim 3, wherein R₄ is optionallysubstitued aryl or aralkyl.
 5. The compound of claim 4, wherein R₄includes at least on hydroxyl, thio, or optionally substitutedloweralkyloxy, arloxy, heteroaryloxy, loweralkylthio, arylthio,heteroarylthio, loweralkylcarbonyl, arylcarbonyl, or heteroarylcaronbylmoiety.
 6. The compound of claim 5, wherein R₄ is seleced from the groupconsisting of phenyl, phenyloxyloweralkyl, and phenylloweralkyl.
 7. Thecompound of claim 6, wherein R₄ is further substitued optionally with amoiety selected from the group consisting of halogen, loweralkyl,haloloweralkyl, loweralkyloxy, halolowerlakloxy, carboxy,loweralkyloxcarbonyl, aryloxycaronyl, (cycloloweralkyl)oxycarbonyl,aralkyloxycarbonyl, heteroaryloxycarbonyl, heteroaralkyloxycarbony,(heterocyloloweralkyl)oxycarbonyl; loweralkylsulfinyl,loweralkylsulfonyl, loweralkythio, aylthio, loweralkylcarbonyloxy,arylcarbonyloxy, aralkylcarbonyloxy, heteroarlycarbonyloxy,heteroaralkylcarbonyloxy, (cycloloweralkyl)carbonyloxy,(heterocycloloweralkyl)carbonyloxy, aminocarbonyl,loweralkylaminocarbonyl, arylaminocarbonyl, aralkylaminocarbonyl,heteroarylaminocarbonyl, cyano, nitro, amino, loweralkylamino, andheteroaralkylaminocarbonyl.
 8. The compound of claim 1 wherein n i s2and each X₁₀ is seleceted independently from the group consisting ofnitrogen, optionally substitued nitrogen, optionally substituedmethylene, and optionally substitued methine.
 9. The compound of claim8, wherein R₄ is selected from the group consisting of optionallysubstituted aryl, heteraryl, aralkyl, and heteroaralkyl.
 10. Thecompound of claim 9, wherein R₄ is optionally substituted aryl oraralkyl.
 11. The compound of claim 10, wherein R₄ includes at least onhydroxyl, thio, or optionally substituted loweralkyloxy, aryloxy,heteroaryloxy, loweralkylthio, arylthio, heteroarylthio,loweralkylcarbonyl, arylcarbonyl, or heteroarylcarbonyl moiety.
 12. Thecompound of claim 11, wherein R₄ is selected from the group consistingof phenyl, phenyloxyloweralkyl, and phenylloweralkyl.
 13. The compoundof claim 12, wherein R₄ is further substituted optionally with a moietyselected from the group consisting of halogen, loweralkyl,halolowerlalkyl, loweralkyloxy, halolowerlakyloxy, carboxy,loweralkyloxycarbonyl, aryloxcarbonyl, (cycloweralkyl)oxycarbonyl,aralkyloxcarbonyl, heteroaryloxcarbonyl, heteroaralkyloxycarbony,(heterocycloloweralkyl)oxycarbonyl, loweralkylsulfinyl,loweralkylsulfonyl, loweralkylthio, arylthio, loweralkylcarbonyloxy,arylcarbonyloxy, aralkylcarbonyloxy, heterarylcarbonyloxy,heteroaralkylcarbonyloxy, (cycloloweralkyl)carbonyloxy,(heterocycloweralkyl)carbonyloxy, aminocarbonyl,loweralkylaminocarbonyl, arylaminocarbonyl, aralkylamainocarbonyl,heteroarylaminocarbonyl, cyano, nitro, amino, loweralkylamino, andheteroaralkylaminocarbonyl.
 14. The compound of claim 1, wherein X₆-X₉are selected independently from the group consisting of nitrogen andoptionally substituted methine.
 15. The compound of claim 14, wherein atleast one of X₆-X₉ is methine substituted with a moiety selected fromthe group consisting of loweralkyloxy, aryloxy, heteroaryloxy,loweralkylthio, arylthio, heteroarylthio, loweralkylcarbony,arylcarbonyl, and heteroarylcarbonyl.
 16. The compound of claim 15,wherein X₇ is methine substitued with hydroxy or loweralkyloxy.
 17. Thecompound of claim 14, wherein n is 1 and X₁₀ is selected from the groupconsisting of nitrogen, optionally substituted nitrogen, and optionallysubstitued methylene or methine.
 18. The compound of claim 17, whereinR₄ is selected from the group consisting of optionally substituted aryl,heteroaryl, aralkyl, and heteroaralkyl.
 19. The compound of claim 18,wherein R₄ is optionally substituted aryl or aralkyl.
 20. The compoundof claim 19, wherein R₄ includes at least on hydroxyl, thio, oroptionally substituted loweralkyloxy, arloxy, heteroaryloxy,loweralkylthio, arylthio, heteroarylthio, loweralkylcarbonyl,arylcarbonyl, or heteroarylcarbonyl moiety.
 21. The compound of claim20, wherein R₄ is selected from the group consisting of phenyl,phenloxyloweralkyl, and phenylloweralkyl.
 22. The compound of claim 21,wherein R₄ is further substituted optionally with a moiety selected fromthe group consisting of halogen, loweralkyl, halowerlalkyl,loweralkyloxy, halolowerlakloxy, carboxy, loweralkyloxcarbonyl,aryloxcarbonyl, (cycloweralkyl)oxycarbonyl, aralkyloxycarbonyl,heteroaryloxcarbonyl, heteraralkyloxycarbonly(heterocycloloweralkyl)oxycarbonyl, loweralkylsulfinyl,loweralkylsufonyl, loweralkythio, arylthio, loweralkylcarbonyloxy,arylcarbonyloxy, aralkycarbonyloxy, heteroarylcarbonyloxy,heteroaralkylcarbonyloxy, (cycloloweralkyl)carbonyloxy,(heterocycloloweralkyl)carbonyloxy, aminocarbonyl,loweralkylaminocarbonyl, arylaminocarbonyl, aralylaminocarbonyl,heterarylaminocarbonyl, cyano, nitro, amino, loweralkylamino, andheteroaralkylaminocarbonyl.
 23. The compound of claim 14, wherein n is 2and each X₁₀ is selected independently from the group consisting ofnitrogen, optinally substituted nitrogen, optionally substituedmethylene, and optionally substituted methine.
 24. The compound of claim23, wherein R₄ is selected from the group consisting of optionallysubstituted aryl, heteroaryl, aralkyl, and heteroaralkyl.
 25. Thecompound of claim 24, wherein R₄ is optionally substitued aryl oraralkyl.
 26. The compound of claim 25, wherein R₄ includes at least onehydroxyl, thio, or optionally substituted loweralkyloxy, arloxy,heteroaryloxy, loweralkythio, arylthio, heteroarylthio,loweralkycarbonyl, arylcarbonyl, or heteroarylcarbonyl moiety.
 27. Thecompound of claim 26, wherein R₄ is selected from the group consitsingof phenyl, phenyloxyloweralkyl, and phenylloweralkyl.
 28. The compoundsof claim 27, wherein R₄ is further substitued optionally with a moietyselected from the group consisting of halogen, loweralkyl,halolowelalkyl, loweralkyloxy, halolowerlakyloxy, carboxy,loweralkyloxcarbonyl, aryloxcarbonyl, (cycloweralkyl)oxycarbonyl,aralkyloxycarbonyl, heteroaryloxycarbonyl, heteroaralkyloxycarbonyl(heterocycloloweralkyl)oxycarbonyl, loweralkylsulfinyl,loweralkylsuflonyl, loweralkylthio, arylthio, loweralkylcarbonloxy,arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy,heteroaralkycarbonyloxy, (cycloloweraalkyl)carbonyloxy,(heterocycloloweralkyl)carbonyloxy, aminocarbonyl,loweraklylaminocarbony, arylaminocarbonyl, aralkylaminocarbonyl,heteroarylaminocarbonyl, cyano, nitro, amino, loweralkylamino, andheteroaralkylaminocarbonyl.
 29. A composition for use in treating anestrogen receptor-mediated disorder in a mammal, comprising atherapeutically effective amount of a compound of claim 1 in apharamaceutically effective carrier.
 30. A method for treating anestrogen receptor-mediated disorder in a mammal, comprisingadministering to such mammal a therapeutically effective amount of acompound of claim 1 in a pharmaceutically effective carrier.
 31. Themethod of claim 30, wherein said disease is chosen from the groupconsisting of: osteoporosis, estrogen-dependent cancer, andestrogen-dependent illness.
 32. A method for preventing an estrogenreceptor-mediated disorder in a mammal, comprising administering to such mammal a prophylactically effective amount of a compound of claim 1in a pharmaceutically effective carrier.
 33. The method of claim 32,wherein said disease is chosen from the group consisting of:osteoporosis, estrogen-dependent cancer, and estrogen-dependent illnes..34. A method for modulating the biological activity of an estrogenreceptor, comprising exposign said estrogen receptor to a compound ofcalim 1, to modulate thereby the binding of said estrogen reeptor to anassociated estrogen receptor elemetn.
 35. The method of claim 34,wherein said estrgen receptor is the α isoform.
 36. The method of claim34, wherein said estrogen receptor is the β isoform.